Novel adenovirus gene therapy vehicle and cell line

ABSTRACT

A novel adenovirus E1/E4 expressing packaging cell line is provided, which permits the generation of recombinant adenoviruses deleted in both gene regions. The E1/E4 deleted recombinant adenovirus is capable of expressing a selected transgene product in cells in vivo or in vitro. This recombinant virus is useful in the treatment of genetic disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation of U.S. patent application Ser. No.09/650,594, filed Aug. 30, 2000, which is a continuation of U.S. patentapplication Ser. No. 08/549,489, filed Oct. 27, 1995, now U.S. Pat. No.6,281,010, which is a continuation-in-part of U.S. patent applicationSer. No. 08/462,014, filed Jun. 5, 1995, now U.S. Pat. No. 5,756,283.The disclosure of parent application Ser. No. 08/462,014 is incorporatedby reference herein.

[0002] This invention was supported by the National Institute of HealthGrant Nos. HD32649-01 and DK49136. The United States government hasrights in this invention.

BACKGROUND OF THE INVENTION

[0003] The present invention relates to the field of somatic genetherapy and the treatment of genetic disorders.

[0004] Adenoviruses are eukaryotic DNA viruses that can be modified toefficiently deliver a therapeutic or reporter transgene to a variety ofcell types. Human adenoviruses are comprised of a linear, approximately36 kb double-stranded DNA genome, which is divided into 100 map units(m.u.), each of which is 360 bp in length. The DNA contains shortinverted terminal repeats (ITR) at each end of the genome that arerequired for viral DNA replication. The gene products are organized intoearly (E1 through E4) and late (L1 through L5) regions, based onexpression before or after the initiation of viral DNA synthesis [see,e.g., M. S. Horwitz et al, “Adenoviridae and Their Replication”,Virology, second edition, pp. 1712, ed. B. N. Fields et al, Raven PressLtd., New York (1990)]. The adenoviruses types 2 and 5 (Ad2 and Ad5,respectively), are not associated with human malignancies.

[0005] Recombinant adenoviruses are capable of providing extremely highlevels of transgene delivery to virtually all cell types, regardless ofthe mitotic state. The efficacy of this system in delivering atherapeutic transgene in vivo that complements a genetic imbalance hasbeen demonstrated in animal models of various disorders [K. F. Kozarskyet al, Somatic Cell Mol. Genet., 19:449-458 (1993) (“Kozarsky et al I”);K. F. Kozarsky et al, J. Biol. Chem., 269:13695-13702 (1994) (“Kozarskyet al II); Y. Watanabe, Atherosclerosis, 36:261-268 (1986); K. Tanzawaet al, FEBS Letters, 118(1):81-84 (1980); J. L. Golasten et al, NewEngl. J. Med., 309(11983) :288-296 (1983); S. Ishibashi et al, J. Clin.Invest., 92:883-893 (1993); and S. Ishibashi et al, J. Clin. Invest.,93:1885-1893 (1994)]. The use of recombinant adenoviruses in thetransduction of genes into hepatocytes in vivo has previously beendemonstrated in rodents and rabbits [see, e.g., Kozarsky II, citedabove, and S. Ishibashi et al, J. Clin. Invest., 92:883-893 (1993)].

[0006] The first-generation recombinant, replication-deficientadenoviruses which have been developed for gene therapy containdeletions of the entire E1a and part of the E1b regions. Thisreplication-defective virus is grown on an adenovirus-transformed,complementation human embryonic kidney cell line containing a functionaladenovirus E1a gene which provides a transacting E1a protein, the 293cell [ATCC CRL1573]. E1-deleted viruses are capable of replicating andproducing infectious virus in the 293 cells, which provide E1a and E1bregion gene products in trans. The resulting virus is capable ofinfecting many cell types and can express the introduced gene (providingit carries its own promoter), but cannot replicate in a cell that doesnot carry the El region DNA unless the cell is infected at a very highmultiplicity of infection.

[0007] However, in vivo studies revealed transgene expression in theseE1 deleted vectors was transient and invariably associated with thedevelopment of severe inflammation at the site of vector targeting [S.Ishibashi et al, J. Clin. Invest., 93:1885-1893 (1994); J. M. Wilson etal, Proc. Natl. Acad. Sci., USA, 85:4421- 4424 (1988); J. M. Wilson etal, Clin. Bio., 3:21-26 (1991); M. Grossman et al, Som. Cell. and Mol.Gen., 17:601-607 (1991)]. Antigenic targets for immune mediatedclearance are viral proteins expressed from the recombinant viral genomeand/or the product of the transgene [Y. Yang et al, Proc. Natl. Acad.Sci., USA, 91:4407-4411 (May 1994); Y. Yang et al, Immun., 1:433-442(August 1994)].

[0008] There remains a need in the art for additional recombinantadenoviruses, therapeutic compositions and methods which enableeffective treatment of disorders and diseases by gene therapy.

SUMMARY OF THE INVENTION

[0009] In one aspect of this invention, a novel packaging cell line isprovided which expresses adenovirus genes E1a, E1b and E4, or functionalfragments thereof. In one embodiment, the E4 gene fragment is openreading frame (ORF) 6 under the control of an inducible promoter.

[0010] In another aspect, the invention provides a recombinantadenovirus comprising the DNA of, or corresponding to, at least aportion of the genome of an adenovirus having functional deletions ofthe E1 and E4 gene regions; a suitable gene operatively linked toregulatory sequences directing its expression, and an adenovirus capsid,the recombinant virus capable of infecting a mammalian cell andexpressing the gene product in the cell in vivo or in vitro. In apreferred embodiment, the cell is a muscle cell.

[0011] In another aspect, the invention provides a mammalian cellinfected with the recombinant virus described above.

[0012] In still another aspect, the invention provides a recombinantadenovirus shuttle vector comprising the DNA of, or corresponding to, atleast a portion of the genome of an adenovirus having functionaldeletions of the E1 and E4 gene regions; a suitable gene operativelylinked to regulatory sequences capable of directing its expression; andplasmid sequences.

[0013] In still a further aspect, the invention provides a method fordelivering and stably integrating a selected gene into a mammalian cellcomprising introducing into said cell an effective amount of arecombinant virus described above.

[0014] In another aspect, the invention provides a method for producingthe above-described recombinant Ad virus by co-transfecting the shuttlevector described above and a helper adenovirus into the packaging cellline described above, wherein the transfected cell generates therecombinant adenovirus. The recombinant adenovirus is subsequentlyisolated and purified therefrom.

[0015] Other aspects and advantages of the present invention aredescribed further in the following detailed description of the preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 is a schematic drawing of an exemplary plasmid useful forthe construction of a packaging cell line of this invention. PlasmidpMMTVE4ORF6 or pMTE4ORF6, which contains a mouse mammary tumor viruspromoter (MMTV promoter) or a sheep metallothionine promoter (MTpromoter), respectively, in control of a human E4 ORF 6 gene sequence, agrowth hormone gene terminator sequence (GH), an SV40 origin ofreplication, plasmid sequences from a pBR322-based plasmid including aneomycin resistance gene, an SV40 polyadenylation site and an ampicillinresistance gene.

[0017]FIGS. 2A through 2F provides the continuous DNA sequence [SEQ IDNO: 1] of the minigene containing the MMTV promoter in operative controlof the adenovirus serotype 5 E4 gene open reading frame 6. Nucleotides1-1506 provide the MMTV promoter. Nucleotides 1523-2408 span E4 ORF6 andthe amino acid sequence of ORF 6 [SEQ ID NO: 2] is indicated under theORF DNA sequence. Nucleotides 2409-3654 span the growth hormone gene(GH) terminator sequences, which provide the polyadenylation site.

[0018]FIG. 3 is a schematic map of recombinant adenovirus H5.001CBLacZ,with indicated restriction endonuclease enzyme sites. The striated barrepresents the CBLacZ minigene; the black bar represents Ad5 viralbackbone, the crosshatched bar represents Ad E4 deletion.

[0019] FIGS. 4A through 4CC provide the DNA sequence [SEQ ID NO: 3] ofrecombinant adenovirus H5.001CBLacZ in which nucleotides 1-330 span Admap units 0-1; nucleotides 370-928 span the CMV enhancer/chicken β-actinpromoter (CB);nucleotides 945-4429 encode E. coli β-galactosidase,nucleotides 4429-4628 span the polyadenylation sequence; and nucleotides4671-35408 span Ad5 sequences m.u. 9.2 to about m.u. 92.1 and from aboutm.u. 97.3 to m.u. 100 (containing a substantial deletion of the E4 genebetween m.u. 92 through 97.2).

[0020]FIG. 5 is a graph plotting LacZ forming units/ml vs time (hours)for E4 complementing cell lines infected with H5.001CBLacZ.

[0021]FIG. 6A is a graph of the induction, ORF6 expression and viralproduction in 293-27-18 packaging cells plotting yield at 24 hourspost-infection in LacZ forming units (LFU)/ml and ORF6 protein (abs.mm)vs. concentration of the inducer, dexamethasone (μM). The unitreference, abs.mm, indicates the intensity of the size of the proteinband on a Western blot and reflects absorbence and protein size inmm^(2.) The square represents the yield at 24 hours post infection. Thediamond represents ORF6 protein detected at 24 hours post-infection.

[0022]FIG. 6B is a graph of the induction, ORF6 expression and viralproduction in 293-10-3 packaging cells plotting yield at 24 hourspost-infection in LFU/ml and ORF6 protein (abs.mm) vs. concentration ofthe inducer, zinc (μM). The symbols are as described for FIG. 6A.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention provides novel packaging cell lines, whichenable the production of recombinant adenoviruses functionally deletedin both the E1 and E4 genes, and methods which enable the therapeutictreatment of disorders with such recombinant adenoviruses.

[0024] To increase the transgene capacity and decrease immune responseof recombinant adenoviral vectors, as many viral genes as possibleshould be deleted to inactivate the adenovirus. However, it is crucialto generate complementing cell lines for construction and propagation ofsuch deleted adenoviral vectors. The method and compositions of thepresent invention overcome several problems previously identified in thegene therapy for first generation E1 deleted adenoviruses and displayadvantages in administration particularly to muscle tissue.

I. Novel Packaging Cell Lines

[0025] Early region 4 (E4) of adenovirus serotype 5 consists of 7 openreading frames (ORFs) believed to be involved in viral DNA replication,host cell shut-off, and late mRNA accumulation. To generate recombinantadenoviruses (Ad) deleted in E4, the function of the E4 region must besupplied to the recombinant virus by a helper virus or packaging cellline. However, useful packaging cell lines have not been availablebecause normally the continuous expression of functioning Ad E1 andfunctional E4 in a single cell line are toxic to the cell. Such cellsare therefore not useful for the growth and replication of recombinantadenoviruses. Further, the DNA encoding the functional Ad E1 and Ad E4genes, when present in a packaging cell line, can increase the chancesof recombination with a recombinant Ad virus to cause the virus torevert to a wildtype Ad virus.

[0026] The present invention avoids these problems by providing apackaging cell line which contains the Ad5 E1 gene and only the ORF 6 ofthe Ad5 E4 gene. ORF6 of E4 alone can provide the requirements for E4 inthe viral life cycle. According to this invention, the ORF6 is furtherpreferably under the transcriptional control of an inducible promoter,such as the sheep metallothionine promoter, inducible by zinc, or themouse mammary tumor virus (MMTV) promoter, inducible by aglucocorticoid, particularly, dexamethasone. This packaging cell linepermits one to control the development of toxicity by regulating theexpression of the E4 ORF6 gene. After the desired shuttle vectorcontaining the adenoviral sequences is transfected into the cell line,expression of the E4 ORF6 can be induced by the appropriate inducer. Thepackaging cell is thus able to provide both Ad E1 and Ad E4 ORF6 geneproducts to the recombinant virus for a sufficient period to allowproductive infection and recovery of the recombinant virus, before thecell becomes toxic. At present, the time period before the cellexperiences toxicity is about 10 days.

[0027] In its most preferred form, the packaging cell line is a humanembryonic kidney (HEK) 293 E1 expressing cell line into which isintroduced the E4 ORF 6 sequence under the control of an induciblepromoter. The MMTV promoter with its glucocorticoid inducer is presentlypreferred, because the zinc sulfate inducer of the MT promoter canitself be toxic to the cells. However, other inducible promoters, suchas those identified in International patent application WO95/13392,published May 18, 1995, and incorporated by reference herein may also beused in the production of packaging cell lines according to thisinvention. Constitutive promoters in control of the expression of ORF6may be employed, such as the constitutive Ad5 E4 region promoter, LTR,but are less preferred.

[0028] It should be understood by one of skill in the art that anotherparent cell line may be selected for the generation of a novel cell lineexpressing the E1a, E1b, and E4 ORF6 genes of a selected adenovirusserotype. Among such parent cell lines may be included HeLa [CCL 2],A549 [CCL 185], KB [CCL 17], Detroit [e.g., Detroit 510, CCL 72] andWI-38 [ATCC CCL 75] cells. These cell lines are all available from theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209. Other suitable parent cell lines may be obtained fromother sources. If such parent cell lines were selected for modification,the cell line would need to be further supplied with the E1a and E1bgene functions, e.g., such as by transfection with a plasmid containingthese genes or functional fragments thereof under a suitable promoter,as well as with the ORF6 gene as described herein.

[0029] Example 1 below provides specific teaching of the construction ofpackaging cell lines containing only the ORF 6 of Ad5 E4 region or, forfunctional comparisons, the entire E4 region. Briefly described, theentire E4 region and an ORF6 sequence of Ad 5 E4 gene were obtained byknown techniques [see, e.g., Sambrook et al., “Molecular Cloning. ALaboratory Manual.”, 2d edit., Cold Spring Harbor Laboratory, New York(1989) and references cited therein]. To isolate the ORF6 region, theanchored polymerase chain reaction technique was used to amplify theORF6 sequence from its initiation codon to its termination codon.Primers selected from the published sequence of ORF6 were used toamplify the ORF sequence and insert restriction sites onto the end ofthe sequence. The E4 ORF6 sequence itself is reproduced as nucleotides1523 through 2408 of SEQ ID NO: 1 in FIG. 2. The entire E4 gene sequenceis published in the Genbank sequence of Ad5 [Genbank Accession No.M73260].

[0030] A minigene was constructed that placed the ORF6 sequence underthe control of a selected promoter. By “minigene” as used here is meantthe combination of the ORF6 sequence and the other regulatory elementsnecessary to transcribe the sequence and express the gene product in acell containing that minigene. The ORF6 sequence gene is operativelylinked to regulatory components in a manner which permits itstranscription. Such components include conventional regulatory elements,such as a promoter to drive ORF6 expression. One inducible promoter wasan Zn⁺² inducible sheep metallothionine (MT) promoter [M. G. Peterson etal, Eur. J. Biochem., 174:417-424 (1988)]. The second promoter, i.e, thepromoter exemplified in FIG. 2, is the dexamethasone-inducible mousemammary tumor virus (MMTV) promoter. The DNA sequence of the MMTVpromoter spans nucleotides 1-1506 of SEQ ID NO: 1 in FIG. 2.

[0031] The minigene also contains nucleic acid sequences heterologous tothe ORF6 viral sequence, including sequences providing signals requiredfor efficient polyadenylation of the transcript (poly-A or pA). A commonpoly-A sequence which is employed in this invention is that derived fromthe growth hormone (GH) gene terminator sequence. The poly-A sequencegenerally is inserted in the minigene following the ORF6 sequence. ThepolyA sequence employed in the MMTV-ORF6 minigene described in Example 1and FIG. 2 is supplied by the growth hormone gene terminator, whichspans nucleotides 2409-3654 of SEQ ID NO: 1 in FIG. 2 and an SV40 originof replication. A similar minigene differing in promoter sequence, polyAsequence and/or SV40 origin of replication sequence can also be designedby one of skill in the art to transfer the E4 ORF6 sequence to a shuttleplasmid. Selection of these and other common vector elements areconventional [see, e.g., Sambrook et al, “Molecular Cloning. ALaboratory Manual.”, 2d edit., Cold Spring Harbor Laboratory, New York(1989) and references cited therein] and many such sequences areavailable from commercial and industrial sources as well as fromGenbank.

[0032] The ORF6-containing minigene was subcloned into a pBR322-basedshuttle plasmid that contained a neomycin resistance gene, resulting inthe shuttle vector depicted in FIG. 1. Any of the many known bacterialshuttle vectors may be employed to carry the minigene, providing thatthe vector contains a reporter gene or selectable marker of which many,e.g., neo, amp or purimycin, are known in the art. It is expected thatone of skill in the art can develop other suitable shuttle vectors usingother plasmid components which are similarly capable of transferring theORF6 minigene into the chromosome of a cell transfected with theplasmid.

[0033] As further described in Example 1, other shuttle vectors weredesigned for comparative purposes, which contain the complete orsubstantially complete Ad5 E4 region under the control of theconstitutive retroviral MLV LTR sequence in the presence or absence ofthe endogenous E4 promoter. The shuttle plasmid carrying the ORF6minigene (or the entire E4 region) was introduced into HEK 293 cellswhich express the Ad E1 gene products. Complementing cell lines weregenerated that express these Ad E4 or ORF6 genes from either theirendogenous promoters or heterologous inducible promoters. These celllines are further characterized by their genetic constitution, E4protein synthesis, recombinant AAV helper function, relative plaqueefficiency of H5dl1004 virus, and growth kinetics of recombinant E1/E4deleted adenovirus. These characteristics of exemplary E1/E4 expressingpackaging cell lines are discussed in detail in the following examples.

[0034] The E1/E4 ORF6 expressing packaging cell lines are useful in thegeneration of recombinant E1/E4 deleted adenoviruses. These recombinantadenoviruses are useful in transferring a selected transgene to aselected cell. In in vivo experiments with the recombinant virus grownin the packaging cell lines, the E1/E4 deleted recombinant virusdemonstrated utility particularly in transferring a transgene to amuscle cell.

II. Recombinant Adenovirus

[0035] The novel E1/E4 expressing cell line is useful in furtherconstructing E1/E4 deleted recombinant adenoviruses containing anyselected transgene. The recombinant adenoviruses of this invention arecapable of delivering a suitable gene to mammalian cells and tissues.These recombinant adenoviruses are functionally deleted in at least theE1a, E1b and E4 Ad gene regions. By the term “functionally deleted” ismeant that a sufficient amount of the gene region is removed orotherwise damaged, e.g., by mutation or modification, so that the generegion is no longer capable of producing the products of geneexpression. If desired, the entire gene region may be removed.

[0036] The adenovirus sequences used in the construction of the shuttlevectors, helper viruses, if needed, and recombinant viruses, and othercomponents and sequences employed in the construction of the vectors andviruses described herein may be readily obtained from commercial oracademic sources based on previously published and described sequences.Viral materials may also be obtained from an individual patient. Theviral sequences and vector components may be generated by resort to theteachings and references contained herein, coupled with standardrecombinant molecular cloning techniques known and practiced by thoseskilled in the art. Modifications of existing nucleic acid sequencesforming the vectors, including sequence deletions, insertions, and othermutations taught by this specification may be generated using standardtechniques. Similarly, the methods employed for the selection of viralsequences useful in a vector, the cloning and construction of the“minigene” and its insertion into a desired viral shuttle vector and theproduction of a recombinant infectious virus are within the skill in theart given the teachings provided herein.

A. Construction of the Transgene containing “Minigene”

[0037] A “minigene” in this context is defined as above, except that thecomponents of this minigene are designed to express the gene product invivo. Such components include conventional regulatory elements necessaryto drive expression of the transgene in a cell transfected with therecombinant virus. For this minigene, a selected promoter is operativelylinked to the transgene and located, with other regulatory elements,within the selected viral sequences of the recombinant vector. Selectionof the promoter is a routine matter and is not a limitation of thisinvention. Useful promoters may be constitutive promoters or regulated(inducible) promoters, which will enable control of the amount of thetransgene to be expressed. For example, a desirable promoter is that ofthe cytomegalovirus (CMV) immediate early promoter/enhancer [see, e.g.,Boshart et al, Cell, 41:521-530 (1985)]. Another desirable promoterincludes the Rous sarcoma virus LTR promoter/enhancer. Still anotherpromoter/enhancer sequence is the chicken cytoplasmic β-actin (CB)promoter [T. A. Kost et al, Nucl. Acids Res., 11(23) :8287 (1983)].Other suitable promoters may be selected by one of skill in the art.

[0038] The minigene may also desirably contain nucleic acid sequencesheterologous to the viral vector sequences including poly-A sequencesand introns with functional splice donor and acceptor sites, asdescribed above. The poly-A sequence generally is inserted in theminigene following the transgene sequences and before the 3′ adenovirussequences. A minigene of the present invention may also contain anintron, desirably located between the promoter/enhancer sequence and thetransgene. Selection of these and other common vector elements areconventional as described above and many such sequences are availablefrom commercial and industrial sources as well as from Genbank.

[0039] As above stated, the minigene is located in the site of anyselected deletion in the recombinant adenovirus. In the exemplary E1/E4deleted recombinant adenovirus H5.001CBLacZ, the transgene is located inthe deleted E1 gene region. However, the transgene may be locatedelsewhere in the adenovirus sequence, as desired.

B. Production of Recombinant Adenovirus

[0040] Adenovirus sequences useful in this invention may include the DNAsequences of a number of adenovirus types, which are available fromGenbank, including type Ad5 [Genbank Accession No. M73260]. Theadenovirus sequences may be obtained from any known adenovirus serotype,such as serotypes 2, 3, 4, 7, 12 and 40, and further including any ofthe presently identified 41 human types [see, e.g., Horwitz, citedabove]. Similarly, adenoviruses known to infect other animals may alsobe employed in the vector constructs of this invention. The selection ofthe adenovirus type is not anticipated to limit the following invention.A variety of adenovirus strains are available from the American TypeCulture Collection, Manassas, Virginia, or available by request from avariety of commercial and institutional sources. In the followingexemplary embodiment an adenovirus, type 5 (Ad5) is used forconvenience.

[0041] However, it is desirable to obtain a variety of adenovirusshuttle vectors based on different human adenovirus serotypes. It isanticipated that a library of such plasmids and the resultingrecombinant adenoviruses would be useful in a therapeutic regimen toevade cellular, and possibly humoral, immunity, and lengthen theduration of transgene expression, as well as improve the success ofrepeat therapeutic treatments. Additionally the use of various serotypesis believed to produce recombinant viruses with different tissuetargeting specificities. Additionally, the absence of adenoviral genesE1 and E4 in the recombinant adenovirus of this invention should reduceor eliminate adverse CTL responses which normally cause destruction ofrecombinant adenoviruses deleted of only the E1 gene.

[0042] Recombinant adenoviruses of this invention are recombinant,defective adenoviruses (i.e., E1 deleted) which are also deletedcompletely or functionally of the E4 gene region. Functional deletionsof E4 gene regions may be assessed by assays of Examples 2 and 3, amongother assays. Recombinant adenoviruses of useful in this invention mayoptionally bear other mutations, e.g., temperature sensitive mutationsin the E2a gene region, and deletions in the E3 gene regions.

[0043] An adenovirus of this invention contains a functional deletion ofthe adenoviral early immediate early gene E1a (which spans mu 1.3 to4.5) and delayed early gene E1b (which spans mu 4.6 to 11.2). Similarlythe adenovirus has a functional deletion of the E4 region (which spansmu 92 to 97.2), or at least of ORF6 of the E4 region.

[0044] Gene regions which may be optionally deleted in the E1/E4 deletedrecombinant viruses of this invention include all or a portion of theadenovirus delayed early gene E3 (which spans mu 76.6 to 86.2). Thefunction of E3 is irrelevant to the function and production of therecombinant virus particle.

[0045] The recombinant adenovirus of this invention may also have amutation which results in reduced expression of adenoviral proteinand/or reduced viral replication. For example, a temperature-sensitivemutation may be introduced into the adenovirus delayed early gene E2a(which spans mu 67.9 to 61.5). Among such mutations include theincorporation of the missense temperature-sensitive (ts) mutation in the(DBP) E2a region found in the Ad5 H5ts125 strain [P. Vander Vliet et al,J. Virol., 15:348-354 (1975)] at 62.5 mu. A single amino acidsubstitution (62.5 mu) at the carboxy end of the 72 kd protein producedfrom the E2a gene in this strain produces a protein product which is asingle-stranded DNA binding protein and is involved in the replicationof adenoviral genomic DNA. At permissive temperatures (approximately 32°C. ) the ts strain is capable of full life cycle growth on HeLa cells,while at non-permissive temperatures (approximately 38° C. ) noreplication of adenoviral DNA is seen. In addition, at non-permissivetemperatures, decreased immunoreactive 72 kd protein is seen in HeLacells. See, e.g., J. F. Engelhardt et al, Hum. Gene Ther., 5:1217-1229(1994); J. F. Engelhardt et al, Proc. Natl. Acad. Sci., USA,91:6196-6200 (1994) and International patent application Ser. No.WO95/13392, published May 18, 1995, incorporated by reference herein.

[0046] However, it should be understood that other deletions in theadenovirus genome as previously described in the art or otherwise mayalso occur in the recombinant viruses of this invention. One minimaltype of recombinant adenovirus can contain adenovirus genomic sequencesfrom which all viral genes are deleted. More specifically, theadenovirus sequences may be only the cis-acting 5′ and 3′ invertedterminal repeat (ITR) sequences of an adenovirus (which function asorigins of replication) and the native 5′ packaging/enhancer domain,that contains sequences necessary for packaging linear Ad genomes andenhancer elements for the E1 promoter. The adenovirus 5′ sequencecontaining the 5′ ITR and packaging/enhancer region (Ad5 mu 0-1 or bp1-360) can be employed as the 5′ adenovirus sequence in recombinantadenoviruses of this invention. The 3′ adenovirus sequences includingthe right terminal (3′) ITR sequence of the adenoviral genome spanningabout bp 35,353—end of the adenovirus genome, or map units ^(˜)98.4-100may be desirably employed as the 3′ sequence of the recombinantadenovirus. These sequences, which are clearly devoid of the E1 and E4genes, can flank, or be operatively associated with the minigene in arecombinant virus. Any other necessary Ad gene products will then besupplied by helper viruses and the E1/E4 ORF6 expressing packaging cellof this invention.

[0047] Exemplary recombinant adenoviruses for use in this invention, forexample, may be obtained by homologous recombination of desiredfragments from various recombinant adenoviruses, a technique which hasbeen commonly employed to generate other recombinant adenoviruses forgene therapy use. In the examples below, a representative recombinantadenovirus, H5.001CBLacZ, is constructed by homologous recombinationbetween the adenovirus dl1004 (also H5dl1004) viral backbone andpAdCBLacZ minigene DNA. H5dl1004 is an Ad5 virus deleted of from aboutmap unit 92.1 through map unit 98, i.e, substantially the entire E4gene. The dl1004 virus is described in Bridge and Ketner, J. Virol.,632(2) :631-638 (Feb. 1989), incorporated by reference herein.

[0048] The pAdCBLacZ vector is a cDNA plasmid containing Ad m.u. 0-1, anE1 deletion into which is inserted a bacterial β-galactosidase geneunder the control of a chicken β-actin promoter, with other regulatoryelements as described below, and flanked by Ad m.u. 9-16 and plasmidsequence.

[0049] The production of the E1/E4 recombinant adenovirus of thisinvention in the novel packaging cell line of this invention utilizesconventional techniques. Such techniques include conventional cloningtechniques of cDNA such as those described in texts [Sambrook et al,cited above], use of overlapping oligonucleotide sequences of theadenovirus genomes, polymerase chain reaction, and any suitable methodwhich provides the desired nucleotide sequence. Standard transfectionand co-transfection techniques are employed, e.g., CaPO₄ transfectiontechniques using the complementation 293 cell line. Other conventionalmethods employed include homologous recombination of the viral genomes,plaquing of viruses in agar overlay, methods of measuring signalgeneration, and the like.

[0050] For example, following the construction and assembly of thedesired minigene-containing plasmid vector pAdCBLacZ, the E1/E4expressing packaging cell line of this invention is infected with thehelper virus H5dl1004. The infected cell line is then subsequentlytransfected with the an adenovirus plasmid vector by conventionalmethods. Homologous recombination occurs between the E4-deleted H5dl1004helper and the pAdCBLacZ vector, which permits the adenovirus-transgenesequences in the vector to be replicated and packaged into virioncapsids, resulting in the recombinant virus. About 30 or more hourspost-transfection, the cells are harvested, an extract prepared and therecombinant virus containing the LacZ transgene is purified by buoyantdensity ultracentrifugation in a CsCl gradient.

III. Use of the Recombinant Virus in Gene Therapy

[0051] The resulting recombinant adenovirus containing the transgeneproduced by cooperation of the adenovirus vector and E4 deleted helpervirus and packaging cell line, as described above, thus provides anefficient gene transfer vehicle which can deliver the transgene to apatient in vivo or ex vivo and provide for integration of the gene intoa mammalian cell.

[0052] The above-described recombinant viruses are administered tohumans in a conventional manner for gene therapy and serve as analternative or supplemental gene therapy for the disorder to which thetransgene is directed. A recombinant adenovirus of this invention may beadministered to a patient, preferably suspended in a biologicallycompatible solution or pharmaceutically acceptable delivery vehicle. Asuitable vehicle includes sterile saline. Other aqueous and non-aqueousisotonic sterile injection solutions and aqueous and non-aqueous sterilesuspensions known to be pharmaceutically acceptable carriers and wellknown to those of skill in the art may be employed for this purpose.

[0053] The recombinant viruses are administered in sufficient amounts totransfect the desired target cells, e.g., muscle, liver, epithelial,etc. and provide sufficient levels of transfer and expression of thetransgene to provide a therapeutic benefit without undue adverse or withmedically acceptable physiological effects which can be determined bythose skilled in the medical arts. Conventional and pharmaceuticallyacceptable routes of administration include direct delivery to themuscle or other selected cell, intranasal, intravenous, intramuscular,subcutaneous, intradermal, oral and other parental routes ofadministration. Routes of administration may be combined, if desired.

[0054] Dosages of recombinant virus will depend primarily on factorssuch as the condition being treated, the age, weight and health of thepatient, and may thus vary among patients. For example, atherapeutically effective human dose of the recombinant adenovirus isgenerally in the range of from about 20 to about 100 ml of salinesolution containing concentrations of from about 1×10⁹ to 1×10¹¹ pfu/mlvirus. A preferred human dose is estimated to be about 50 ml salinesolution at 2×10¹⁰ pfu/ml. The dose will be adjusted to balance thetherapeutic benefit against any side effects. The levels of expressionof the transgene can be monitored to determine the frequency ofadministration.

[0055] An optional method step involves the co-administration to thepatient, either concurrently with, or before or after administration ofthe recombinant virus of a suitable amount of a short acting immunemodulator. The selected immune modulator is defined herein as an agentcapable of inhibiting the formation of neutralizing antibodies directedagainst the recombinant vector of this invention or capable ofinhibiting or substantially delaying cytolytic T lymphocyte (CTL)elimination of the vector. Among desirable immune modulators areinterleukin-12 [European Patent Application No. 441,900]; gammainterferon [S. C. Morris et al, J. Immunol., 152:1047 (1994)];interleukin-4 [U.S. Pat. No. 5,017,691]; antibody to the CD4 protein,such as anti-OKT 3+ [see, e.g., U.S. Pat. No. 4,658,019] or antibodyGK1.5 (ATCC Accession No. TIB207); a soluble CD40 molecule or anantibody to CD40 ligand (Bristol-Myers Squibb Co) [European patentapplication 555,880, published Aug. 18, 1993]; a soluble form of B7 oran antibody to CD28 or CTLA4 [CTLA4-Ig (Bristol-Myers Squibb Co),European patent application 606,217, published Jul. 20, 1994], or agentssuch as cyclosporin A or cyclophosphamide.

[0056] Thus, the compositions and methods of this invention provide adesirable gene therapy treatment.

[0057] The following examples illustrate the construction and testing ofthe novel packaging cell lines, the E1/E4 deleted recombinant adenovirusof the present invention and the use thereof. These examples areillustrative only, and do not limit the scope of the present invention.

EXAMPLE 1 Novel E1a/E1b and E4 Expressing Packaging Cell Lines A.Construction of E4 ORF 6 Expressing Plasmids

[0058] 1. pMTE4ORF6

[0059] One exemplary plasmid useful for the construction of a packagingcell line of this invention is pMTE4ORF6, which contains a sheepmetallothionine promoter (MT promoter) [M. G. Peterson et al, citedabove] in control of the transcription of a human E4 ORF 6 gene sequence(nucleotides 1521 to 2406 of SEQ ID NO: 1 in FIG. 2), a growth hormoneterminator (GH), an SV40 origin of replication, plasmid sequences frompBR322-based plasmid including a neomycin resistance gene, an SV40polyadenylation site and an ampicillin resistance gene.

[0060] The various functional fragments of this plasmid may be readilyreplaced with other conventionally used sequences and are not criticalto the design of the plasmid.

[0061] 2. pMMTVE4ORF6

[0062] Another exemplary plasmid useful for the construction of apackaging cell line of this invention is pMMTVE4ORF6, which contains amouse mammary tumor virus promoter (MMTV) (nucleotides 1-1506 of SEQ IDNO:1 in FIG. 2) in transcriptional control of a human E4 ORF 6 genesequence (nucleotides 1523-2408 of SEQ ID NO: 1 in FIG. 2), a growthhormone terminator (GH) (nucleotides 2409-3654 of SEQ ID NO: 1 in FIG.2), an SV40 origin of replication, plasmid sequences from plasmidpBR322, including a neomycin resistance gene, and an ampicillinresistance gene. The various functional fragments of this plasmid may bereadily replaced with other conventionally used sequences and are notcritical to the design of the plasmid.

[0063] 3. pLTR.E4(−) Endogenous E4 Promoter

[0064] A plasmid used as a control for the construction of a packagingcell line of this invention is pLTR.E4(−). This plasmid contains theconstitutive retroviral MLV LTR and most of the Ad E4 gene region exceptthat the endogenous E4 promoter and a portion of E4 ORF1 are missing.The other plasmid sequences remain the same as described above.

[0065] 4. pLTR.E4(+) Endogenous E4 Promoter

[0066] Still another plasmid useful for the study of the methods of thisinvention is pLTR.E4, which contains the constitutive MLV LTR andendogenous E4 promoter and an intact E4 gene. The other plasmidsequences remain the same as described above.

B. Transfections and Selection of Clones

[0067] Each of the above-described plasmids was transfected by thecalcium phosphate precipitation technique into the human embryonickidney cell line 293 [ATCC CRL1573] which expresses the product of theadenovirus E1 genes, seeded on 100 mm plates (10 μg plasmid/plate).Twenty four hours post-transfection, cells were harvested and seeded atvarying dilutions (1:10-1:100) in 100 mm plates for about 10 days.Seeding media contain G418 (Geneticin, BRL) at 1 mg/ml. Resistantcolonies that developed were selected using the following assays andexpanded. Preliminary analysis of clones was based on enhancedtransduction efficiency of a recombinant adeno-associated virus,AV.CMVLacZ, and immunofluorescence localization of Ad E4 protein asdescribed in the following examples.

EXAMPLE 2 AV-CMVLacZ Transduction Enhancement Assay

[0068] E1 and E4 Ad gene products are needed for recombinantadeno-associated virus (AAV) function. This primary assay involvesseeding the packaging cell lines of Example 1 in 96 well 35 mm cultureplates (2×10⁶ cells/well) and infecting the cells with purified,heat-treated AV.CMVLacZ at an MOI of 1000 virus particles/cell.

A. Preparation of AV.CMVLacZ

[0069] A recombinant AAV virus was prepared by conventional geneticengineering techniques for the purposes of this experiment. RecombinantAAV was generated by plasmid transfections in the presence of helperadenovirus [Samulski et al, J. Virol., 63:3822-3828 (1989)]. Acis-acting plasmid pAV.CMVLacZ was derived from psub201 [Samulski et al,J. Virol., 61:3096-3101 (1987)] and contains an E. coil β galactosidaseminigene in place of AAV Rep and Cap genes. The 5′ to 3′ organization ofthe recombinant AV.CMVLacZ genome (4.9 kb) includes

[0070] (a) the 5′ AAV ITR (bp 1-173) was obtained by PCR using pAV2 [C.A. Laughlin et al, Gene, 23: 65-73 (1983)] as template;

[0071] (b) a CMV immediate early enhancer/promoter [Boshart et al, Cell,41:521-530 (1985)];

[0072] (c) an SV40 intron;

[0073] (d) E. coli beta-galactosidase cDNA;

[0074] (e) an SV40 polyadenylation signal (a 237 Bam HI-BclI restrictionfragment containing the cleavage/poly-A signals from both the early andlate transcription units; and

[0075] (f) 3′ AAV ITR, obtained from pAV2 as a SnaBI-BglII fragment.

[0076] Rep and Cap genes were provided by a trans-acting plasmid pAAV/Ad[Samulski et al, cited above].

[0077] Monolayers of 293 cells grown to 90% confluency in 150 mm culturedishes (5×10⁷ cells/plate) were infected with H5.CBALP at an MOI of 10.H5.CBALP (also called H5.010ALP) is a recombinant adenovirus thatcontains an alkaline phosphatase minigene in place of adenovirus E1a andE1b gene sequences (map units 1-9.2 of the Ad5 sequence of GenBank[Accession No. M73260]). The alkaline phosphatase cDNA is under thetranscriptional control of a CMV-enhanced β-actin promoter in thisvirus. This helper virus is described in Goldman et al, Hum. Gene Ther.,6:839-851 (July, 1995); Engelhardt et al, Hum. Gene Ther., 5:1217-1229(October, 1994); and references cited therein.

[0078] Infections were done in Dulbecco's Modified Eagles Media (DMEM)supplemented with 2% fetal bovine serum (FBS) at 20 ml media/150 mmplate. Two hours post-infection, 50 μg plasmid DNA (37.5 μg trans-actingand 12.5 μg cis-acting) in 2.5 ml of transfection cocktail was added toeach plate and evenly distributed. Transfections were calcium phosphatebased as described [B. Cullen, Meth. Enzymol., 152:684-704 (1987)].Cells were left in this condition for 10-14 hours after which theinfection/transfection media was replaced with 20 ml fresh DMEM/2% FBS.Forty to fifty hours post-transfection, cells were harvested, suspendedin 10 mM Tris-Cl (pH 8.0) buffer (0.5 ml/150 mm plate) and a lysateprepared by sonication. The lysate was brought to 10 mM manganesechloride, after which bovine pancreatic DNase I (20,000 units) and RNase(0.2 mg/ml final concentration) were added, and the reaction incubatedat 37° C. for 30 minutes. Sodium deoxycholate was added to a finalconcentration of 1% and incubated at 37° C. for an additional 10minutes.

[0079] The treated lysate was chilled on ice for 10 minutes and solidCsCl added to a final density of 1.3 g/ml. The lysate was brought to afinal volume of 60 ml with 1.3 g/ml CsCl solution in 10 mM Tris-Cl (pH8.0) and divided into three equal aliquots. Each 20 ml sample waslayered onto a CsCl step gradient composed of two 9.0 ml tiers withdensities 1.45 g/ml and 1.60 g/ml.

[0080] Centrifugation was performed at 25,000 rpm in a Beckman SW-28rotor for 24 hours at 4° C.

[0081] Fractions containing peak titers of functional AV.CMVLacZ viruswere combined and subjected to three sequential rounds of equilibriumsedimentation in CsCl. Rotor selection included a Beckman NVT-90 (80,000rpm for 4 hours) and SW-41 (35,000 rpm for 20 hours). At equilibrium,AV.CMVLacZ appeared as an opalescent band at 1.40-1.41 g/ml CsCl.Densities were calculated from refractive index measurements. Purifiedvector was exchanged to 20 mM HEPES buffer (pH7.8) containing 150 mMNaCl (HBS) by dialysis and stored frozen at −80° C. in the presence of10% glycerol or as a liquid stock at −20° C. in HBS/40% glycerol.

[0082] Purified virus was tested for contaminating H5.CBALP helper virusand AV.CMVLacZ titers. Helper virus was monitored by histochemicalstaining for reporter alkaline phosphatase activity. A sample ofpurified virus representing 1.0% of the final product was added to agrowing monolayer of 293 cells seeded in a 60 mm plate. Forty-eighthours later, cells were fixed in 0.5% glutaraldehyde/phosphate bufferedsaline (PBS) for 10 minutes at room temperature, washed in PBS (3×10minutes) and incubated at 65° C. for 40 minutes to inactivate endogenousalkaline phosphatase activity. The monolayer was allowed to cool to roomtemperature, rinsed once briefly in 100 mM Tris-Cl (pH9.5)/100 mM NaCl/5mM MgCl, and incubated at 37° C. for 30 minutes in the same buffercontaining 0.33 mg/ml nitroblue tetrazolium chloride (NBT) and 0.165mg/ml 5-bromo-4-choro-3-indolphosphate p-toluidine salt (BCIP). Colordevelopment was stopped by washing the monolayer in 10 mM Tris-Cl (pH8.0)/5 mM EDTA. Routinely the purification scheme described aboveremoved all detectable H5.CBALP helper virus by the third round ofbuoyant density ultracentrifugation.

[0083] AV.CMVLacZ titers were measured according to genome copy number(virus particles/ml), absorbance at 260 nm (A₂₆₀ particles/ml) and LacZForming Units (LFU/ml). Virus particle concentrations were based onSouthern blotting. Briefly, a sample of purified AV.CMVLacZ was treatedwith capsid digestion buffer (50 mM Tris-Cl, pH 8.0/1.0 mM EDTA, pH8.0/0.5% SDS/Proteinase K 1.0 mg/ml) at 50° C. for one hour to releasevirus DNA. The reactions were allowed to cool to room temperature,loading dye was added and electrophoresed through a 1.2% agarose gel.Standard quantities of ds AV.CMVLacZ genome were also resolved on thegel.

[0084] DNAs were electroblotted onto a nylon membrane, hybridized with a³²P random primer labeled restriction fragment, and the resulting blotscanned on a PhosphorImager 445 SI (Molecular Dynamics). A standardcurve was generated from the duplex forms and used to extrapolate thenumber of virus genomes in the sample. LFU titers were generated byinfecting indicator cells with limiting dilutions of virus sample.Indicator cells included HeLa and 293. Twenty-four hours later, cellswere fixed in glutaraldehyde and cells were histochemically stained forE. coli β-galactosidase (LacZ) activity as described in J. M. Wilson etal, Proc. Natl. Acad. Sci. USA, 85:3014-3018 (1988). One LFU isdescribed as the quantity of virus that is sufficient to cause visuallydetectable β-galactosidase expression in one cell 24 hourspost-infection.

B. Induction of ORF6 Expression

[0085] Induction of ORF6 expression with 10 μM dexamethasone or 150 μMzinc sulfate (for negative control, no inducer used) was initiated 2hours before the addition of virus and continued throughout the durationof the experiment. Twenty-four hours after the addition of virus, cellswere harvested, lysates were generated by sonication and analyzed forthe β-galactosidase expression (i.e., β-galactosidase activity) andvirus DNA as described above. Hirt extracts were prepared from lowmolecular weight DNA from cell extracts. The preparation of the Hirtextracts and subsequent analysis by Southern hybridization wereperformed by resort to conventional procedures known to one of skill inthe art.

[0086] In the absence of the inducers, the packaging cell lines generatelower levels of β-galactosidase in rAAV infected cells. Induction ofORF6 expression with the inducer dexamethasone results in a concomitantrise in AV.CMVLacZ cell transduction to a level that was much greaterthan the parent 293 line. Expression of E1 alone was insufficient tohave an effect in the adenovirus mediated augmentation of rAAVtransduction.

[0087] Results are demonstrated for certain positive clones in the TableI below (see Example 4). However, for 30 cell lines having an MMTVpromoter and ORF6 sequence, 4 demonstrated over 90% blue cellsillustrative of LacZ production in the presence of dexamethasone, i.e.,293-27-6, 293-27-17, 293-27-18 and 293-27-28.

EXAMPLE 3 Immunofluorescence Localization of Ad5 Late Protein

[0088] Positive clones from the assay of Example 2 were infected withthe recombinant E4 deleted adenovirus H5dl1004 and screened for E4complementation using an immunofluorescence assay for late geneexpression. The H5dl1004 virus was obtained from Dr. Ketner of JohnsHopkins University and is described in Bridge and Ketner, J. Virol.,632(2) :631-638 (Feb. 1989), incorporated by reference herein. BecauseORF6 of E4 complements late Ad gene expression, specifically in theformation of the hexon and penton fibers of the adenovirus, cell linescontaining ORF6 are able to bind with antibody against these proteins.

[0089] Each cell line of Example 1 is infected with E4 deleted virusH5dl1004 virus at an MOI of 0.1. The cells were treated with mouseanti-adenovirus FITC-labeled monoclonal antibody to either the hexon orpenton fibers in a 1:10 dilution (Chemicon International Inc., Temecula,Calif. ). Positive clones were identified by reaction with the antibody.

EXAMPLE 4 Relative Plaguing Efficiency

[0090] The cell lines of Example 1 demonstrating with strongcomplementation ability in Example 3 were screened for relative plaquingefficiency of H5dl1004 as compared to W162 cells (an E4-complementingVero cell line which does not express E1) [Weinberg and Ketner, Proc.Natl. Acad. Sci, USA, 80(17) :5383-5386 (1983)]. In Table II below,RPE%, i.e., relative plaguing efficiency, represents the titer ofH5dl1004 on tested cell lines/titer of H5dl1004 on W162 cells. Forexample, the RPE of 293 cells is 0.

[0091] The positive cell lines selected by all criteria are identifiedin Table I below, with the results of the assays of Examples 2, 3 and 4.TABLE I E1/E4 Double Complementing Cell Lines Cell Trans- Pro- AV.CMVLine Gene moter IF/LP LacZ RPE % 293-10-3  ORF6 MT ++++ ++++ 246293-39-11 ORF6 LTR ++++ +++ 52 293-84-31 E4- LTR ++++ ++++ 179 293-12-31whole LTR + ++++ ++++ 174 E4 E4 293-27-6  ORF6 MMTV +++++ 327 293-27-17ORF6 MMTV ++++ 313 293-27-18 ORF6 MMTV +++++ 339 293-27-28 ORF6 MMTV++++ 261

EXAMPLE 5 Construction and Purification of H5.001CBLacZ

[0092] The plasmid pAd.CBLacZ was constructed as described in detail inK. Kozarsky et al, Som. Cell Mol. Genet., 19(5): 449-458 (1993),incorporated by reference herein. This plasmid contained a minigenecomprising a 5′ flanking NheI restriction site, followed by Ad5 sequencem.u. 0-1, followed by an E1 deletion into which is inserted a CMVenhancer/chicken β-actin promoter sequence [T. A. Kost et al, Nucl.Acids Res., 11(23) :8287 (1983)], which controls the transcription ofthe following bacterial β-galactosidase, followed by a poly A sequenceand flanked 3′ by Ad m.u. 9-16, and another NheI site. In the plasmid,the minigene was flanked on both sides by plasmid sequence containingdrug resistance markers.

[0093] The plasmid pAd.CBLacZ was linearized with NheI andco-transfected by the calcium phosphate co-transfection method into thenovel packaging cell line of Example 1 with ClaI digested H5dl1004 (anAd5 sequence deleted of from about map unit 92.1 through map unit 98,corresponding to substantially the entire E4 gene).

[0094] Homologous recombination occurs in the cell line between thesetwo viral constructs between Ad map units 9-16, resulting in recombinantadenovirus, designated H5.001CBLacZ (FIGS. 3 and 4 ). This recombinantadenovirus contains the sequence from about nucleotide 1 to about 4628from pAd.CBLacZ and Ad5 map units 9-92.1 and 97.3 to 100 from H5dl1004.This recombinant adenovirus is thereby functionally deleted, andsubstantially structurally deleted, of the Ad E1 and E4 genes.

[0095] Viral plaques were selected and screened by the β-galactosidaseassay [Wilson (1988), cited above] and H5.001CBLacZ was isolatedfollowing three rounds of plaque purification. The purified virus wasalso subjected to cesium chloride density centrifugation and large scaleproduction. For the following mouse experiments, virus was used aftercolumn purification and glycerol was added to a final concentration of10% (v/v). Virus was stored at −70° C. until use.

EXAMPLE 6 Growth Kinetics of H5.001CBLacZ in Packaging Cell Lines

[0096] The cell lines reported in Example 1 were infected withrecombinant H5.001CBLacZ at an MOI of 0.5. The growth kinetics of thisvirus in the E4 complementing cell lines are shown in FIG. 5.

[0097] Maximum viral yield is reported as LFU/ml in Table II below.TABLE II Cell Line Maximum Viral Yield 293-10-3  2.8 × 10¹⁰ 293-39-119.5 × 10⁸  293-84-31 1.1 × 10⁹  293-12-31 4.5 × 10⁸  293-27-6  2.8 ×10¹⁰ 293-27-17 2.5 × 10¹⁰ 293-27-18 2.9 × 10¹⁰ 293-27-28 1.2 × 10¹⁰

[0098] When grown in 293-27-18 cells (the E4 ORF6 cell line with MMTVpromoter inducible by dexamethasone) the maximum yield of this virus is2.9×10¹⁰ LFU/ml. Several of the cell lines were passaged between 5 and20 times and the viral production of the passages remained stable.However, RPE did fall following repeated passages of cells.

EXAMPLE 7 Other Recombinant Adenoviruses

[0099] Other related recombinant adenoviruses were prepared similarly toH5.001CBLacZ by homologous recombination between pAdCBLacZ and otherhelper viruses.

[0100] As one example, H5.000CBLacZ is a recombinant E1 deleted Ad5which contains the same minigene as H5.001CBLacZ, but has an intact E4gene. This recombinant virus was prepared as described by homologousrecombination between pAdCBLacZ and a wild-type Ad5.

[0101] As another example, H5.010CBLacZ contains the adenovirus mapunits 0-1, followed by a CMV enhanced, chicken cytoplasmic β-actinpromoter, the E. Coli beta-galactosidase gene (lacZ), a polyadenylationsignal (pA), and adenovirus type 5 map units 9-100, with a smalldeletion in the E3 gene (the Ad 5 sub360 backbone). This recombinantvirus may be prepared by homologous recombination between the pAdCBLacZvector and Ad5 virus sub360, which contains a 150 bp deletion within the14.6 kD protein of the E3 gene. See, e.g., J. F. Engelhardt et al, Proc.Natl. Acad. Sci., USA, 91:6196-6200 (June 1994); and Engelhardt et al,Hum. Gene Ther., 5:1217-1229 (Oct. 1994), both incorporated by referenceherein.

[0102] These recombinant adenoviruses were isolated followingtransfection [Graham, Virol., 52:456-467 (1974)], and were subjected totwo rounds of plaque purification. Lysates were purified by cesiumchloride density centrifugation as previously described [Englehardt etal, Proc. Natl. Acad. Sci. USA, 88:11192-11196 (1991)]. Cesium chloridewas removed by passing the virus over a BioRad DG10 column usingphosphate-buffered saline.

EXAMPLE 8 LacZ Gene Transfer into Mouse A. Transfer into Mouse Muscle

[0103] Five to six-week old male C57B/6 mice were anesthetized. Anteriortibialis muscles were exposed and directly injected with eitherrecombinant adenovirus H5.000CBLacZ, H5.010CBLacZ or H5.001CBLacZ asfollows: 25 μL of purified viral suspension at a stock concentration of5×10¹¹ virus particles/mL was injected by inserting the tip of the 33gauge needle of a 100 μL Hamilton syringe into the belly of the muscle.

[0104] Animals were sacrificed on day 4, 14, 28 and 60 post injection.The muscles were dissected and frozen in liquid nitrogen cooledisopentane. Six μM sections were cut in a cryostat, fixed and stainedfor β-galactosidase activity for 6 hours at 37° C.

[0105] While the blue stained recombinant virus was found for each virusin the day 4 and day 14 (most abundant) stains, by day 28, theH5.001CBLacZ clearly demonstrated more virus on day 28. By day 60, theonly virus which stained positive was the H5.001CBLacZ.

B. Transfer into Mouse Lung and Circulation

[0106] Recombinant adenovirus H5.000CBLacZ (control), and H5.001CBLacZ(1×10¹¹ viral particles) were administered to six week old C57BL/6female mice by tail vein injection and trachea installation. The animalswere sacrificed and their liver and lung tissues were harvested at days4, 9, 21, 28 and 35 post-administration. The transgene and viral lategene expression were compared.

[0107] At therapeutic doses of virus, there was diminished expression oflate viral proteins at all time points in comparison with transgene.

C. Dose Responses in Liver

[0108] Dose responses of E4-deleted and E4 intact recombinantadenoviruses in the liver of C57BL/6 mice were studied by tail veinadministration of 1.5×10¹¹, 5×10¹⁰, 1.7×10¹⁰, 5.6×10⁹, and 1.9×10⁹ viralparticles and comparing the transgene and viral late gene expression atday 4, 21, 28, 35, and 42 post administration.

[0109] At therapeutic doses of virus, there was diminished expression oflate viral proteins at all time points in comparison with transgene.

EXAMPLE 9 Other Gene Transfers A. Human OTC Gene Transfer

[0110] The human OTC gene [A. L. Horwich et al, Science, 224:1068-174(1984)] or the human CFTR gene [Riordan et al, Science, 245:1066-1073(1989)] was used to replace the LacZ as the transgene in the recombinantE1/E4 deleted adenoviruses described above, using the techniquesanalogous for the construction of the above-described LacZ vectors.

[0111] The resulting human OTC-containing recombinant viruses wereadministered at an MOI of 10 to 30 to human hepatocytes. The E1/E4deleted recombinant adenovirus demonstrated less replication and lesslate gene expression than when the E1/E4 deleted recombinantadenoviruses are administered to muscle, as described in the exampleabove. However, the results of this gene transfer are better thancomparable transfers with recombinant adenoviruses containing only adeletion in the E1 gene or a deletion in the E1 gene and a pointmutation in the E2a gene.

[0112] Similar results are demonstrated when the transgene is CFTR andthe method of administration is intratracheal into lungs.

[0113] All references recited above are incorporated herein byreference. Numerous modifications and variations of the presentinvention are included in the above-identified specification and areexpected to be obvious to one of skill in the art. Such modificationsand alterations to the compositions and processes of the presentinvention, such as selections of different transgenes and plasmids forthe construction of the packaging cell lines and recombinantadenoviruses, or selection or dosage of the viruses or immunemodulators, are believed to be within the scope of the claims appendedhereto.

1 3 3653 base pairs nucleic acid double not relevant cDNA CDS 1521..24051 CTGCATGTGT CAGAGGTTTT CACCGTCATC ACCGAAACGC GCGAGGCAGC AAGCTTGGCA 60GAAATGGTTG AACTCCCGAG AGTGTCCTAC ACCTAGGGGA GAAGCAGCCA AGGGGTTGTT 120TCCCACCAAG GACGACCCGT CTGCGCACAA ACGGATGAGC CCATCAGACA AAGACATATT 180CATTCTCTGC TGCAAACTTG GCATAGCTCT GCTTTGCCTG GGGCTATTGG GGGAAGTTGC 240GGTTCGTGCT CGCAGGGCTC TCACCCTTGA CTCTTTCAAT AATAACTCTT CTGTGCAAGA 300TTACAATCTA AACAATTCGG AGAACTCGAC CTTCCTCCTG AGGCAAGGAC CACAGCCAAC 360TTCCTCTTAC AAGCCGCATC GATTTTGTCC TTCAGAAATA GAAATAAGAA TGCTTGCTAA 420AAATTATATT TTTACCAATA AGACCAATCC AATAGGTAGA TTATTAGTTA CTATGTTAAG 480AAATGAATCA TTATCTTTTA GTACTATTTT TACTCAAATT CAGAAGTTAG AAATGGGAAT 540AGAAAATAGA AAGAGACGCT CAACCTCAAT TGAAGAACAG GTGCAAGGAC TATTGACCAC 600AGGCCTAGAA GTAAAAAAGG GAAAAAAGAG TGTTTTTGTC AAAATAGGAG ACAGGTGGTG 660GCAACCAGGG ACTTATAGGG GACCTTACAT CTACAGACCA ACAGATGCCC CCTTACCATA 720TACAGGAAGA TATGACTTAA ATTGGGATAG GTGGGTTACA GTCAATGGCT ATAAAGTGTT 780ATATAGATCC CTCCCCTTTC GTGAAAGACT CGCCAGAGCT AGACCTCCTT GGTGTATGTT 840GTCTCAAGAA AAGAAAGACG ACATGAAACA ACAGGTACAT GATTATATTT ATCTAGGAAC 900AGGAATGCAC TTTTGGGGAA AGATTTTCCA TACCAAGGAG GGGACAGTGG CTGGACTAAT 960AGAACATTAT TCTGCAAAAA CTTATGGCAT GAGTTATTAT GATTAGCCTT GATTTGCCCA 1020ACCTTGCGGT TCCCAAGGCT TAAGTAAGTT TTTGGTTACA AACTGTTCTT AAAACAAGGA 1080TGTGAGACAA GTGGTTTCCT GACTTGGTTT GGTATCAAAG GTTCTGATCT GAGCTCTGAG 1140TGTTCTATTT TCCTATGTTC TTTTGGAATT TATCCAAATC TTATGTAAAT GCTTATGTAA 1200ACCAAGATAT AAAAGAGTGC TGATTTTTTG AGTAAACTTG CAACAGTCCT AACATTCACC 1260TCTTGTGTGT TTGTGTCTGT TCGCCATCCC GTCTCCGCTC GTCACTTATC CTTCACTTTC 1320CAGAGGGTCC CCCCGCAGAC CCCGGCGACC CTCAGGTCGG CCGACTGCGG CAGCTGGCGC 1380CCGAACAGGG ACCCTCGGAT AAGTGACCCT TGTCTTTATT TCTACTATTT TGTGTTCGTC 1440TTGTTTTGTC TCTATCTTGT CTGGCTATCA TCACAAGAGC GGAACGGACT CACCTCAGGG 1500AACCAAGCTA GCCCAATTCG ATGACTACGT CCGGCGTTCC ATTTGGCATG ACACTACGAC 1560CAACACGATC TCGGTTGTCT CGGCGCACTC CGTACAGTAG GGATCGTCTA CCTCCTTTTG 1620AGACAGAAAC CCGCGCTACC ATACTGGAGG ATCATCCGCT GCTGCCCGAA TGTAACACTT 1680TGACAATGCA CAACGTGAGT TACGTGCGAG GTCTTCCCTG CAGTGTGGGA TTTACGCTGA 1740TTCAGGAATG GGTTGTTCCC TGGGATATGG TTCTAACGCG GGAGGAGCTT GTAATCCTGA 1800GGAAGTGTAT GCACGTGTGC CTGTGTTGTG CCAACATTGA TATCATGACG AGCATGATGA 1860TCCATGGTTA CGAGTCCTGG GCTCTCCACT GTCATTGTTC CAGTCCCGGT TCCCTGCAGT 1920GTATAGCCGG CGGGCAGGTT TTGGCCAGCT GGTTTAGGAT GGTGGTGGAT GGCGCCATGT 1980TTAATCAGAG GTTTATATGG TACCGGGAGG TGGTGAATTA CAACATGCCA AAAGAGGTAA 2040TGTTTATGTC CAGCGTGTTT ATGAGGGGTC GCCACTTAAT CTACCTGCGC TTGTGGTATG 2100ATGGCCACGT GGGTTCTGTG GTCCCCGCCA TGAGCTTTGG ATACAGCGCC TTGCACTGTG 2160GGATTTTGAA CAATATTGTG GTGCTGTGCT GCAGTTACTG TGCTGATTTA AGTGAGATCA 2220GGGTGCGCTG CTGTGCCCGG AGGACAAGGC GCCTTATGCT GCGGGCGGTG CGAATCATCG 2280CTGAGGAGAC CACTGCCATG TTGTATTCCT GCAGGACGGA GCGGCGGCGG CAGCAGTTTA 2340TTCGCGCGCT GCTGCAGCAC CACCGCCCTA TCCTGATGCA CGATTATGAC TCTACCCCCA 2400TGTAGGGATC CAAGCTTGCG GGCGCATCGA TGATATCAAG CTTGCATGCC TGCAGGTCGA 2460CTCTAGAGGA TCCCGGGTGG NATCCCTGTG ACCCCTCCCC AGTGCCTCTC CTGGCCCTGG 2520AAGTTGGCAC TCCAGTGCCC ACCAGCCTTG TCCTAATAAA ATTAAGTTGN ATCATTTTGT 2580CTGACTAGGT GTCCTTCTAT AATATTATGG GGTGGAGGGG GGTGGTATGG AGCAANGGGN 2640AANTTGGNAA GACAANCTGT AGGGCCTGCG GGGTCTATTG GGAACAAGCT GGAGTGCAGT 2700GGCACAATCT TGGCTCACTG CAATCTCCGC CTCCTGGGTT CAAGCGATTC TCCTGCCTCA 2760GACTCCCGAG TTGTTGGGAT TCCAGGCATG CATGACCAGG CTCAGATAAT TTTTGTTTTT 2820TTGGTAGAGA CGGGGTTTCA CCATATTGGN CAGGCTGGTC TCCAACTCCT AATCTCAGGT 2880GATCTNCCCA CCTTGGCCTC CCAAATTGCT GGGATTACAG GNGTGAACCA CTGNTCCCTT 2940CCCTGTCCTT CTGATTTTAA AATAACTATA CCAGCAGGAG GACGTCCAGA CACAGCATAG 3000GCTACCTGGC CATGCCCAAC CGGTGGGACA TTTGAGTTGC TTGCTTGGCA CTGTCCTCTC 3060ATGCGTTGGG TCCACTCAGT AGATGCCTGT TGAATTGGGT ACGCGGCCAG CTTGGCTGTG 3120GAATGTGTGT CAGTTAGGGT GTGGAAAGTC CCCAGGCTCC CCAGCAGGCA GAAGTATGCA 3180AAGCATGCAT CTCAATTAGT CAGCAACCAG GTGTGGAAAG TCCCCAGGCT CCCCAGCAGG 3240CAGAAGTATG CAAAGCATGC ATCTCAATTA GTCAGNAACC ATAGNCCCGC CCCTAACTCC 3300GTCCATCCCG GCCCTAACTC NGGCCAGTTC CGACCNTNCT CCGGCNNATG GNTGAGTAAT 3360TTGCNNGATT TATGCAGNGG GCGAGGNCGC CTCGGGCTCT GAGNTNTTCC AGAAGTAGTG 3420AGGAGGCTTT NNTGGTGGAA TTGATCAGCT TGGGATCTGA TCAAGAGACA GGATGAGGAT 3480CGNNNCGNAT GATTGAACAA GATGGGTTGC ACGGAGGTTC TCCGGNCGCT TGGGTGGGGA 3540GGNTATTCGG NTATTNTTGG TGNACAACAG NNAAACGGNT GTTCTGATGC CGCCGCGTTC 3600NCGCTTTCAG NGCAGGGGGG CCCCCCTTCT NTTGAGANNA GCNCCCCTTN TTG 3653 294amino acids amino acid <Unknown> not relevant protein 2 Met Thr Thr SerGly Val Pro Phe Gly Met Thr Leu Arg Pro Thr Arg 1 5 10 15 Ser Arg LeuSer Arg Arg Thr Pro Tyr Ser Arg Asp Arg Leu Pro Pro 20 25 30 Phe Glu ThrGlu Thr Arg Ala Thr Ile Leu Glu Asp His Pro Leu Leu 35 40 45 Pro Glu CysAsn Thr Leu Thr Met His Asn Val Ser Tyr Val Arg Gly 50 55 60 Leu Pro CysSer Val Gly Phe Thr Leu Ile Gln Glu Trp Val Val Pro 65 70 75 80 Trp AspMet Val Leu Thr Arg Glu Glu Leu Val Ile Leu Arg Lys Cys 85 90 95 Met HisVal Cys Leu Cys Cys Ala Asn Ile Asp Ile Met Thr Ser Met 100 105 110 MetIle Tyr Gly Tyr Glu Ser Trp Ala Leu His Cys His Cys Ser Ser 115 120 125Pro Gly Ser Leu Gln Cys Ile Ala Gly Gly Gln Val Leu Ala Ser Trp 130 135140 Phe Arg Met Val Val Asp Gly Ala Met Phe Asn Gln Arg Phe Ile Trp 145150 155 160 Tyr Arg Glu Val Val Asn Tyr Asn Met Pro Lys Glu Val Met PheMet 165 170 175 Ser Ser Val Phe Met Arg Gly Arg His Leu Ile Tyr Leu ArgTyr Trp 180 185 190 Tyr Asp Gly His Val Gly Ser Val Val Pro Ala Met SerPhe Gly Tyr 195 200 205 Ser Ala Leu His Cys Gly Ile Leu Asn Asn Ile ValVal Leu Cys Cys 210 215 220 Ser Tyr Cys Ala Asp Leu Ser Glu Ile Arg ValArg Cys Cys Ala Arg 225 230 235 240 Arg Thr Arg Arg Leu Met Leu Arg AlaVal Arg Ile Ile Ala Glu Glu 245 250 255 Thr Thr Ala Met Leu Tyr Ser CysArg Thr Glu Arg Arg Arg Gln Gln 260 265 270 Phe Ile Arg Ala Leu Leu GlnHis His Arg Pro Ile Leu Met His Asp 275 280 285 Tyr Asp Ser Thr Pro Met290 35408 base pairs nucleic acid not relevant not relevant othernucleic acid 3 CATCATCAAT AATATACCTT ATTTTGGATT GAAGCCAATA TGATAATGAGGGGGTGGAGT 60 TTGTGACGTG GCGCGGGGCG TGGGAACGGG GCGGGTGACG TAGTAGTGTGGCGGAAGTGT 120 GATGTTGCAA GTGTGGCGGA ACACATGTAA GCGACGGATG TGGCAAAAGTGACGTTTTTG 180 GTGTGCGCCG GTGTACACAG GAAGTGACAA TTTTCGCGCG GTTTTAGGCGGATGTTGTAG 240 TAAATTTGGG CGTAACCGAG TAAGATTTGG CCATTTTCGC GGGAAAACTGAATAAGAGGA 300 AGTGAAATCT GAATAATTTT GTGTTACTCA TAGCGCGTAA TATTTGTCTAGGGAGATCAG 360 CCTGCAGGTC GTTACATAAC TTACGGTAAA TGGCCCGCCT GGCTGACCGCCCAACGACCC 420 CCGCCCATTG ACGTCAATAA TGACGTATGT TCCCATAGTA ACGCCAATAGGGACTTTCCA 480 TTGACGTCAA TGGGTGGAGT ATTTACGGTA AACTGCCCAC TTGGCAGTACATCAAGTGTA 540 TCATATGCCA AGTACGCCCC CTATTGACGT CAATGACGGT AAATGGCCCGCCTGGCATTA 600 TGCCCAGTAC ATGACCTTAT GGGACTTTCC TACTTGGCAG TACATCTACTCGAGGCCACG 660 TTCTGCTTCA CTCTCCCCAT CTCCCCCCCC TCCCCACCCC CAATTTTGTATTTATTTATT 720 TTTTAATTAT TTTGTGCAGC GATGGGGGCG GGGGGGGGGG GGGGGCGCGCGCCAGGCGGG 780 GCGGGGCGGG GCGAGGGGCG GGGCGGGGCG AGGCGGAGAG GTGCGGCGGCAGCCAATCAG 840 AGCGGCGCGC TCCGAAAGTT TCCTTTTATG GCGAGGCGGC GGCGGCGGCGGCCCTATAAA 900 AAGCGAAGCG CGCGGCGGGC GGGAGCGGGA TCAGCCACCG CGGTGGCGGCCGCAATTCCC 960 GGGGATCGAA AGAGCCTGCT AAAGCAAAAA AGAAGTCACC ATGTCGTTTACTTTGACCAA 1020 CAAGAACGTG ATTTTCGTTG CCGGTCTGGG AGGCATTGGT CTGGACACCAGCAAGGAGCT 1080 GCTCAAGCGC GATCCCGTCG TTTTACAACG TCGTGACTGG GAAAACCCTGGCGTTACCCA 1140 ACTTAATCGC CTTGCAGCAC ATCCCCCTTT CGCCAGCTGG CGTAATAGCGAAGAGGCCCG 1200 CACCGATCGC CCTTCCCAAC AGTTGCGCAG CCTGAATGGC GAATGGCGCTTTGCCTGGTT 1260 TCCGGCACCA GAAGCGGTGC CGGAAAGCTG GCTGGAGTGC GATCTTCCTGAGGCCGATAC 1320 TGTCGTCGTC CCCTCAAACT GGCAGATGCA CGGTTACGAT GCGCCCATCTACACCAACGT 1380 AACCTATCCC ATTACGGTCA ATCCGCCGTT TGTTCCCACG GAGAATCCGACGGGTTGTTA 1440 CTCGCTCACA TTTAATGTTG ATGAAAGCTG GCTACAGGAA GGCCAGACGCGAATTATTTT 1500 TGATGGCGTT AACTCGGCGT TTCATCTGTG GTGCAACGGG CGCTGGGTCGGTTACGGCCA 1560 GGACAGTCGT TTGCCGTCTG AATTTGACCT GAGCGCATTT TTACGCGCCGGAGAAAACCG 1620 CCTCGCGGTG ATGGTGCTGC GTTGGAGTGA CGGCAGTTAT CTGGAAGATCAGGATATGTG 1680 GCGGATGAGC GGCATTTTCC GTGACGTCTC GTTGCTGCAT AAACCGACTACACAAATCAG 1740 CGATTTCCAT GTTGCCACTC GCTTTAATGA TGATTTCAGC CGCGCTGTACTGGAGGCTGA 1800 AGTTCAGATG TGCGGCGAGT TGCGTGACTA CCTACGGGTA ACAGTTTCTTTATGGCAGGG 1860 TGAAACGCAG GTCGCCAGCG GCACCGCGCC TTTCGGCGGT GAAATTATCGATGAGCGTGG 1920 TGGTTATGCC GATCGCGTCA CACTACGTCT GAACGTCGAA AACCCGAAACTGTGGAGCGC 1980 CGAAATCCCG AATCTCTATC GTGCGGTGGT TGAACTGCAC ACCGCCGACGGCACGCTGAT 2040 TGAAGCAGAA GCCTGCGATG TCGGTTTCCG CGAGGTGCGG ATTGAAAATGGTCTGCTGCT 2100 GCTGAACGGC AAGCCGTTGC TGATTCGAGG CGTTAACCGT CACGAGCATCATCCTCTGCA 2160 TGGTCAGGTC ATGGATGAGC AGACGATGGT GCAGGATATC CTGCTGATGAAGCAGAACAA 2220 CTTTAACGCC GTGCGCTGTT CGCATTATCC GAACCATCCG CTGTGGTACACGCTGTGCGA 2280 CCGCTACGGC CTGTATGTGG TGGATGAAGC CAATATTGAA ACCCACGGCATGGTGCCAAT 2340 GAATCGTCTG ACCGATGATC CGCGCTGGCT ACCGGCGATG AGCGAACGCGTAACGCGAAT 2400 GGTGCAGCGC GATCGTAATC ACCCGAGTGT GATCATCTGG TCGCTGGGGAATGAATCAGG 2460 CCACGGCGCT AATCACGACG CGCTGTATCG CTGGATCAAA TCTGTCGATCCTTCCCGCCC 2520 GGTGCAGTAT GAAGGCGGCG GAGCCGACAC CACGGCCACC GATATTATTTGCCCGATGTA 2580 CGCGCGCGTG GATGAAGACC AGCCCTTCCC GGCTGTGCCG AAATGGTCCATCAAAAAATG 2640 GCTTTCGCTA CCTGGAGAGA CGCGCCCGCT GATCCTTTGC GAATACGCCCACGCGATGGG 2700 TAACAGTCTT GGCGGTTTCG CTAAATACTG GCAGGCGTTT CGTCAGTATCCCCGTTTACA 2760 GGGCGGCTTC GTCTGGGACT GGGTGGATCA GTCGCTGATT AAATATGATGAAAACGGCAA 2820 CCCGTGGTCG GCTTACGGCG GTGATTTTGG CGATACGCCG AACGATCGCCAGTTCTGTAT 2880 GAACGGTCTG GTCTTTGCCG ACCGCACGCC GCATCCAGCG CTGACGGAAGCAAAACACCA 2940 GCAGCAGTTT TTCCAGTTCC GTTTATCCGG GCAAACCATC GAAGTGACCAGCGAATACCT 3000 GTTCCGTCAT AGCGATAACG AGCTCCTGCA CTGGATGGTG GCGCTGGATGGTAAGCCGCT 3060 GGCAAGCGGT GAAGTGCCTC TGGATGTCGC TCCACAAGGT AAACAGTTGATTGAACTGCC 3120 TGAACTACCG CAGCCGGAGA GCGCCGGGCA ACTCTGGCTC ACAGTACGCGTAGTGCAACC 3180 GAACGCGACC GCATGGTCAG AAGCCGGGCA CATCAGCGCC TGGCAGCAGTGGCGTCTGGC 3240 GGAAAACCTC AGTGTGACGC TCCCCGCCGC GTCCCACGCC ATCCCGCATCTGACCACCAG 3300 CGAAATGGAT TTTTGCATCG AGCTGGGTAA TAAGCGTTGG CAATTTAACCGCCAGTCAGG 3360 CTTTCTTTCA CAGATGTGGA TTGGCGATAA AAAACAACTG CTGACGCCGCTGCGCGATCA 3420 GTTCACCCGT GCACCGCTGG ATAACGACAT TGGCGTAAGT GAAGCGACCCGCATTGACCC 3480 TAACGCCTGG GTCGAACGCT GGAAGGCGGC GGGCCATTAC CAGGCCGAAGCAGCGTTGTT 3540 GCAGTGCACG GCAGATACAC TTGCTGATGC GGTGCTGATT ACGACCGCTCACGCGTGGCA 3600 GCATCAGGGG AAAACCTTAT TTATCAGCCG GAAAACCTAC CGGATTGATGGTAGTGGTCA 3660 AATGGCGATT ACCGTTGATG TTGAAGTGGC GAGCGATACA CCGCATCCGGCGCGGATTGG 3720 CCTGAACTGC CAGCTGGCGC AGGTAGCAGA GCGGGTAAAC TGGCTCGGATTAGGGCCGCA 3780 AGAAAACTAT CCCGACCGCC TTACTGCCGC CTGTTTTGAC CGCTGGGATCTGCCATTGTC 3840 AGACATGTAT ACCCCGTACG TCTTCCCGAG CGAAAACGGT CTGCGCTGCGGGACGCGCGA 3900 ATTGAATTAT GGCCCACACC AGTGGCGCGG CGACTTCCAG TTCAACATCAGCCGCTACAG 3960 TCAACAGCAA CTGATGGAAA CCAGCCATCG CCATCTGCTG CACGCGGAAGAAGGCACATG 4020 GCTGAATATC GACGGTTTCC ATATGGGGAT TGGTGGCGAC GACTCCTGGAGCCCGTCAGT 4080 ATCGGCGGAA TTACAGCTGA GCGCCGGTCG CTACCATTAC CAGTTGGTCTGGTGTCAAAA 4140 ATAATAATAA CCGGGCAGGC CATGTCTGCC CGTATTTCGC GTAAGGAAATCCATTATGTA 4200 CTATTTAAAA AACACAAACT TTTGGATGTT CGGTTTATTC TTTTTCTTTTACTTTTTTAT 4260 CATGGGAGCC TACTTCCCGT TTTTCCCGAT TTGGCTACAT GACATCAACCATATCAGCAA 4320 AAGTGATACG GGTATTATTT TTGCCGCTAT TTCTCTGTTC TCGCTATTATTCCAACCGCT 4380 GTTTGGTCTG CTTTCTGACA AACTCGGCCT CGACTCTAGG CGGCCGCGGGGATCCAGACA 4440 TGATAAGATA CATTGATGAG TTTGGACAAA CCACAACTAG AATGCAGTGAAAAAAATGCT 4500 TTATTTGTGA AATTTGTGAT GCTATTGCTT TATTTGTAAC CATTATAAGCTGCAATAAAC 4560 AAGTTAACAA CAACAATTGC ATTCATTTTA TGTTTCAGGT TCAGGGGGAGGTGTGGGAGG 4620 TTTTTTCGGA TCCTCTAGAG TCGACCTGCA GGCTGATCAG TGGAAGGTGCTGAGGTACGA 4680 TGAGACCCGC ACCAGGTGCA GACCCTGCGA GTGTGGCGGT AAACATATTAGGAACCAGCC 4740 TGTGATGCTG GATGTGACCG AGGAGCTGAG GCCCGATCAC TTGGTGCTGGCCTGCACCCG 4800 CGCTGAGTTT GGCTCTAGCG ATGAAGATAC AGATTGAGGT ACTGAAATGTGTGGGCGTGG 4860 CTTAAGGGTG GGAAAGAATA TATAAGGTGG GGGTCTTATG TAGTTTTGTATCTGTTTTGC 4920 AGCAGCCGCC GCCGCCATGA GCACCAACTC GTTTGATGGA AGCATTGTGAGCTCATATTT 4980 GACAACGCGC ATGCCCCCAT GGGCCGGGGT GCGTCAGAAT GTGATGGGCTCCAGCATTGA 5040 TGGTCGCCCC GTCCTGCCCG CAAACTCTAC TACCTTGACC TACGAGACCGTGTCTGGAAC 5100 GCCGTTGGAG ACTGCAGCCT CCGCCGCCGC TTCAGCCGCT GCAGCCACCGCCCGCGGGAT 5160 TGTGACTGAC TTTGCTTTCC TGAGCCCGCT TGCAAGCAGT GCAGCTTCCCGTTCATCCGC 5220 CCGCGATGAC AAGTTGACGG CTCTTTTGGC ACAATTGGAT TCTTTGACCCGGGAACTTAA 5280 TGTCGTTTCT CAGCAGCTGT TGGATCTGCG CCAGCAGGTT TCTGCCCTGAAGGCTTCCTC 5340 CCCTCCCAAT GCGGTTTAAA ACATAAATAA AAAACCAGAC TCTGTTTGGATTTGGATCAA 5400 GCAAGTGTCT TGCTGTCTTT ATTTAGGGGT TTTGCGCGCG CGGTAGGCCCGGGACCAGCG 5460 GTCTCGGTCG TTGAGGGTCC TGTGTATTTT TTCCAGGACG TGGTAAAGGTGACTCTGGAT 5520 GTTCAGATAC ATGGGCATAA GCCCGTCTCT GGGGTGGAGG TAGCACCACTGCAGAGCTTC 5580 ATGCTGCGGG GTGGTGTTGT AGATGATCCA GTCGTAGCAG GAGCGCTGGGCGTGGTGCCT 5640 AAAAATGTCT TTCAGTAGCA AGCTGATTGC CAGGGGCAGG CCCTTGGTGTAAGTGTTTAC 5700 AAAGCGGTTA AGCTGGGATG GGTGCATACG TGGGGATATG AGATGCATCTTGGACTGTAT 5760 TTTTAGGTTG GCTATGTTCC CAGCCATATC CCTCCGGGGA TTCATGTTGTGCAGAACCAC 5820 CAGCACAGTG TATCCGGTGC ACTTGGGAAA TTTGTCATGT AGCTTAGAAGGAAATGCGTG 5880 GAAGAACTTG GAGACGCCCT TGTGACCTCC AAGATTTTCC ATGCATTCGTCCATAATGAT 5940 GGCAATGGGC CCACGGGCGG CGGCCTGGGC GAAGATATTT CTGGGATCACTAACGTCATA 6000 GTTGTGTTCC AGGATGAGAT CGTCATAGGC CATTTTTACA AAGCGCGGGCGGAGGGTGCC 6060 AGACTGCGGT ATAATGGTTC CATCCGGCCC AGGGGCGTAG TTACCCTCACAGATTTGCAT 6120 TTCCCACGCT TTGAGTTCAG ATGGGGGGAT CATGTCTACC TGCGGGGCGATGAAGAAAAC 6180 GGTTTCCGGG GTAGGGGAGA TCAGCTGGGA AGAAAGCAGG TTCCTGAGCAGCTGCGACTT 6240 ACCGCAGCCG GTGGGCCCGT AAATCACACC TATTACCGGG TGCAACTGGTAGTTAAGAGA 6300 GCTGCAGCTG CCGTCATCCC TGAGCAGGGG GGCCACTTCG TTAAGCATGTCCCTGACTCG 6360 CATGTTTTCC CTGACCAAAT CCGCCAGAAG GCGCTCGCCG CCCAGCGATAGCAGTTCTTG 6420 CAAGGAAGCA AAGTTTTTCA ACGGTTTGAG ACCGTCCGCC GTAGGCATGCTTTTGAGCGT 6480 TTGACCAAGC AGTTCCAGGC GGTCCCACAG CTCGGTCACC TGCTCTACGGCATCTCGATC 6540 CAGCATATCT CCTCGTTTCG CGGGTTGGGG CGGCTTTCGC TGTACGGCAGTAGTCGGTGC 6600 TCGTCCAGAC GGGCCAGGGT CATGTCTTTC CACGGGCGCA GGGTCCTCGTCAGCGTAGTC 6660 TGGGTCACGG TGAAGGGGTG CGCTCCGGGC TGCGCGCTGG CCAGGGTGCGCTTGAGGCTG 6720 GTCCTGCTGG TGCTGAAGCG CTGCCGGTCT TCGCCCTGCG CGTCGGCCAGGTAGCATTTG 6780 ACCATGGTGT CATAGTCCAG CCCCTCCGCG GCGTGGCCCT TGGCGCGCAGCTTGCCCTTG 6840 GAGGAGGCGC CGCACGAGGG GCAGTGCAGA CTTTTGAGGG CGTAGAGCTTGGGCGCGAGA 6900 AATACCGATT CCGGGGAGTA GGCATCCGCG CCGCAGGCCC CGCAGACGGTCTCGCATTCC 6960 ACGAGCCAGG TGAGCTCTGG CCGTTCGGGG TCAAAAACCA GGTTTCCCCCATGCTTTTTG 7020 ATGCGTTTCT TACCTCTGGT TTCCATGAGC CGGTGTCCAC GCTCGGTGACGAAAAGGCTG 7080 TCCGTGTCCC CGTATACAGA CTTGAGAGGC CTGTCCTCGA GCGGTGTTCCGCGGTCCTCC 7140 TCGTATAGAA ACTCGGACCA CTCTGAGACA AAGGCTCGCG TCCAGGCCAGCACGAAGGAG 7200 GCTAAGTGGG AGGGGTAGCG GTCGTTGTCC ACTAGGGGGT CCACTCGCTCCAGGGTGTGA 7260 AGACACATGT CGCCCTCTTC GGCATCAAGG AAGGTGATTG GTTTGTAGGTGTAGGCCACG 7320 TGACCGGGTG TTCCTGAAGG GGGGCTATAA AAGGGGGTGG GGGCGCGTTCGTCCTCACTC 7380 TCTTCCGCAT CGCTGTCTGC GAGGGCCAGC TGTTGGGGTG AGTACTCCCTCTGAAAAGCG 7440 GGCATGACTT CTGCGCTAAG ATTGTCAGTT TCCAAAAACG AGGAGGATTTGATATTCACC 7500 TGGCCCGCGG TGATGCCTTT GAGGGTGGCC GCATCCATCT GGTCAGAAAAGACAATCTTT 7560 TTGTTGTCAA GCTTGGTGGC AAACGACCCG TAGAGGGCGT TGGACAGCAACTTGGCGATG 7620 GAGCGCAGGG TTTGGTTTTT GTCGCGATCG GCGCGCTCCT TGGCCGCGATGTTTAGCTGC 7680 ACGTATTCGC GCGCAACGCA CCGCCATTCG GGAAAGACGG TGGTGCGCTCGTCGGGCACC 7740 AGGTGCACGC GCCAACCGCG GTTGTGCAGG GTGACAAGGT CAACGCTGGTGGCTACCTCT 7800 CCGCGTAGGC GCTCGTTGGT CCAGCAGAGG CGGCCGCCCT TGCGCGAGCAGAATGGCGGT 7860 AGGGGGTCTA GCTGCGTCTC GTCCGGGGGG TCTGCGTCCA CGGTAAAGACCCCGGGCAGC 7920 AGGCGCGCGT CGAAGTAGTC TATCTTGCAT CCTTGCAAGT CTAGCGCCTGCTGCCATGCG 7980 CGGGCGGCAA GCGCGCGCTC GTATGGGTTG AGTGGGGGAC CCCATGGCATGGGGTGGGTG 8040 AGCGCGGAGG CGTACATGCC GCAAATGTCG TAAACGTAGA GGGGCTCTCTGAGTATTCCA 8100 AGATATGTAG GGTAGCATCT TCCACCGCGG ATGCTGGCGC GCACGTAATCGTATAGTTCG 8160 TGCGAGGGAG CGAGGAGGTC GGGACCGAGG TTGCTACGGG CGGGCTGCTCTGCTCGGAAG 8220 ACTATCTGCC TGAAGATGGC ATGTGAGTTG GATGATATGG TTGGACGCTGGAAGACGTTG 8280 AAGCTGGCGT CTGTGAGACC TACCGCGTCA CGCACGAAGG AGGCGTAGGAGTCGCGCAGC 8340 TTGTTGACCA GCTCGGCGGT GACCTGCACG TCTAGGGCGC AGTAGTCCAGGGTTTCCTTG 8400 ATGATGTCAT ACTTATCCTG TCCCTTTTTT TTCCACAGCT CGCGGTTGAGGACAAACTCT 8460 TCGCGGTCTT TCCAGTACTC TTGGATCGGA AACCCGTCGG CCTCCGAACGGTAAGAGCCT 8520 AGCATGTAGA ACTGGTTGAC GGCCTGGTAG GCGCAGCATC CCTTTTCTACGGGTAGCGCG 8580 TATGCCTGCG CGGCCTTCCG GAGCGAGGTG TGGGTGAGCG CAAAGGTGTCCCTGACCATG 8640 ACTTTGAGGT ACTGGTATTT GAAGTCAGTG TCGTCGCATC CGCCCTGCTCCCAGAGCAAA 8700 AAGTCCGTGC GCTTTTTGGA ACGCGGATTT GGCAGGGCGA AGGTGACATCGTTGAAGAGT 8760 ATCTTTCCCG CGCGAGGCAT AAAGTTGCGT GTGATGCGGA AGGGTCCCGGCACCTCGGAA 8820 CGGTTGTTAA TTACCTGGGC GGCGAGCACG ATCTCGTCAA AGCCGTTGATGTTGTGGCCC 8880 ACAATGTAAA GTTCCAAGAA GCGCGGGATG CCCTTGATGG AAGGCAATTTTTTAAGTTCC 8940 TCGTAGGTGA GCTCTTCAGG GGAGCTGAGC CCGTGCTCTG AAAGGGCCCAGTCTGCAAGA 9000 TGAGGGTTGG AAGCGACGAA TGAGCTCCAC AGGTCACGGG CCATTAGCATTTGCAGGTGG 9060 TCGCGAAAGG TCCTAAACTG GCGACCTATG GCCATTTTTT CTGGGGTGATGCAGTAGAAG 9120 GTAAGCGGGT CTTGTTCCCA GCGGTCCCAT CCAAGGTTCG CGGCTAGGTCTCGCGCGGCA 9180 GTCACTAGAG GCTCATCTCC GCCGAACTTC ATGACCAGCA TGAAGGGCACGAGCTGCTTC 9240 CCAAAGGCCC CCATCCAAGT ATAGGTCTCT ACATCGTAGG TGACAAAGAGACGCTCGGTG 9300 CGAGGATGCG AGCCGATCGG GAAGAACTGG ATCTCCCGCC ACCAATTGGAGGAGTGGCTA 9360 TTGATGTGGT GAAAGTAGAA GTCCCTGCGA CGGGCCGAAC ACTCGTGCTGGCTTTTGTAA 9420 AAACGTGCGC AGTACTGGCA GCGGTGCACG GGCTGTACAT CCTGCACGAGGTTGACCTGA 9480 CGACCGCGCA CAAGGAAGCA GAGTGGGAAT TTGAGCCCCT CGCCTGGCGGGTTTGGCTGG 9540 TGGTCTTCTA CTTCGGCTGC TTGTCCTTGA CCGTCTGGCT GCTCGAGGGGAGTTACGGTG 9600 GATCGGACCA CCACGCCGCG CGAGCCCAAA GTCCAGATGT CCGCGCGCGGCGGTCGGAGC 9660 TTGATGACAA CATCGCGCAG ATGGGAGCTG TCCATGGTCT GGAGCTCCCGCGGCGTCAGG 9720 TCAGGCGGGA GCTCCTGCAG GTTTACCTCG CATAGACGGG TCAGGGCGCGGGCTAGATCC 9780 AGGTGATACC TAATTTCCAG GGGCTGGTTG GTGGCGGCGT CGATGGCTTGCAAGAGGCCG 9840 CATCCCCGCG GCGCGACTAC GGTACCGCGC GGCGGGCGGT GGGCCGCGGGGGTGTCCTTG 9900 GATGATGCAT CTAAAAGCGG TGACGCGGGC GAGCCCCCGG AGGTAGGGGGGGCTCCGGAC 9960 CCGCCGGGAG AGGGGGCAGG GGCACGTCGG CGCCGCGCGC GGGCAGGAGCTGGTGCTGCG 10020 CGCGTAGGTT GCTGGCGAAC GCGACGACGC GGCGGTTGAT CTCCTGAATCTGGCGCCTCT 10080 GCGTGAAGAC GACGGGCCCG GTGAGCTTGA GCCTGAAAGA GAGTTCGACAGAATCAATTT 10140 CGGTGTCGTT GACGGCGGCC TGGCGCAAAA TCTCCTGCAC GTCTCCTGAGTTGTCTTGAT 10200 AGGCGATCTC GGCCATGAAC TGCTCGATCT CTTCCTCCTG GAGATCTCCGCGTCCGGCTC 10260 GCTCCACGGT GGCGGCGAGG TCGTTGGAAA TGCGGGCCAT GAGCTGCGAGAAGGCGTTGA 10320 GGCCTCCCTC GTTCCAGACG CGGCTGTAGA CCACGCCCCC TTCGGCATCGCGGGCGCGCA 10380 TGACCACCTG CGCGAGATTG AGCTCCACGT GCCGGGCGAA GACGGCGTAGTTTCGCAGGC 10440 GCTGAAAGAG GTAGTTGAGG GTGGTGGCGG TGTGTTCTGC CACGAAGAAGTACATAACCC 10500 AGCGTCGCAA CGTGGATTCG TTGATATCCC CCAAGGCCTC AAGGCGCTCCATGGCCTCGT 10560 AGAAGTCCAC GGCGAAGTTG AAAAACTGGG AGTTGCGCGC CGACACGGTTAACTCCTCCT 10620 CCAGAAGACG GATGAGCTCG GCGACAGTGT CGCGCACCTC GCGCTCAAAGGCTACAGGGG 10680 CCTCTTCTTC TTCTTCAATC TCCTCTTCCA TAAGGGCCTC CCCTTCTTCTTCTTCTGGCG 10740 GCGGTGGGGG AGGGGGGACA CGGCGGCGAC GACGGCGCAC CGGGAGGCGGTCGACAAAGC 10800 GCTCGATCAT CTCCCCGCGG CGACGGCGCA TGGTCTCGGT GACGGCGCGGCCGTTCTCGC 10860 GGGGGCGCAG TTGGAAGACG CCGCCCGTCA TGTCCCGGTT ATGGGTTGGCGGGGGGCTGC 10920 CATGCGGCAG GGATACGGCG CTAACGATGC ATCTCAACAA TTGTTGTGTAGGTACTCCGC 10980 CGCCGAGGGA CCTGAGCGAG TCCGCATCGA CCGGATCGGA AAACCTCTCGAGAAAGGCGT 11040 CTAACCAGTC ACAGTCGCAA GGTAGGCTGA GCACCGTGGC GGGCGGCAGCGGGCGGCGGT 11100 CGGGGTTGTT TCTGGCGGAG GTGCTGCTGA TGATGTAATT AAAGTAGGCGGTCTTGAGAC 11160 GGCGGATGGT CGACAGAAGC ACCATGTCCT TGGGTCCGGC CTGCTGAATGCGCAGGCGGT 11220 CGGCCATGCC CCAGGCTTCG TTTTGACATC GGCGCAGGTC TTTGTAGTAGTCTTGCATGA 11280 GCCTTTCTAC CGGCACTTCT TCTTCTCCTT CCTCTTGTCC TGCATCTCTTGCATCTATCG 11340 CTGCGGCGGC GGCGGAGTTT GGCCGTAGGT GGCGCCCTCT TCCTCCCATGCGTGTGACCC 11400 CGAAGCCCCT CATCGGCTGA AGCAGGGCTA GGTCGGCGAC AACGCGCTCGGCTAATATGG 11460 CCTGCTGCAC CTGCGTGAGG GTAGACTGGA AGTCATCCAT GTCCACAAAGCGGTGGTATG 11520 CGCCCGTGTT GATGGTGTAA GTGCAGTTGG CCATAACGGA CCAGTTAACGGTCTGGTGAC 11580 CCGGCTGCGA GAGCTCGGTG TACCTGAGAC GCGAGTAAGC CCTCGAGTCAAATACGTAGT 11640 CGTTGCAAGT CCGCACCAGG TACTGGTATC CCACCAAAAA GTGCGGCGGCGGCTGGCGGT 11700 AGAGGGGCCA GCGTAGGGTG GCCGGGGCTC CGGGGGCGAG ATCTTCCAACATAAGGCGAT 11760 GATATCCGTA GATGTACCTG GACATCCAGG TGATGCCGGC GGCGGTGGTGGAGGCGCGCG 11820 GAAAGTCGCG GACGCGGTTC CAGATGTTGC GCAGCGGCAA AAAGTGCTCCATGGTCGGGA 11880 CGCTCTGGCC GGTCAGGCGC GCGCAATCGT TGACGCTCTA GACCGTGCAAAAGGAGAGCC 11940 TGTAAGCGGG CACTCTTCCG TGGTCTGGTG GATAAATTCG CAAGGGTATCATGGCGGACG 12000 ACCGGGGTTC GAGCCCCGTA TCCGGCCGTC CGCCGTGATC CATGCGGTTACCGCCCGCGT 12060 GTCGAACCCA GGTGTGCGAC GTCAGACAAC GGGGGAGTGC TCCTTTTGGCTTCCTTCCAG 12120 GCGCGGCGGC TGCTGCGCTA GCTTTTTTGG CCACTGGCCG CGCGCAGCGTAAGCGGTTAG 12180 GCTGGAAAGC GAAAGCATTA AGTGGCTCGC TCCCTGTAGC CGGAGGGTTATTTTCCAAGG 12240 GTTGAGTCGC GGGACCCCCG GTTCGAGTCT CGGACCGGCC GGACTGCGGCGAACGGGGGT 12300 TTGCCTCCCC GTCATGCAAG ACCCCGCTTG CAAATTCCTC CGGAAACAGGGACGAGCCCC 12360 TTTTTTGCTT TTCCCAGATG CATCCGGTGC TGCGGCAGAT GCGCCCCCCTCCTCAGCAGC 12420 GGCAAGAGCA AGAGCAGCGG CAGACATGCA GGGCACCCTC CCCTCCTCCTACCGCGTCAG 12480 GAGGGGCGAC ATCCGCGGTT GACGCGGCAG CAGATGGTGA TTACGAACCCCCGCGGCGCC 12540 GGGCCCGGCA CTACCTGGAC TTGGAGGAGG GCGAGGGCCT GGCGCGGCTAGGAGCGCCCT 12600 CTCCTGAGCG GTACCCAAGG GTGCAGCTGA AGCGTGATAC GCGTGAGGCGTACGTGCCGC 12660 GGCAGAACCT GTTTCGCGAC CGCGAGGGAG AGGAGCCCGA GGAGATGCGGGATCGAAAGT 12720 TCCACGCAGG GCGCGAGCTG CGGCATGGCC TGAATCGCGA GCGGTTGCTGCGCGAGGAGG 12780 ACTTTGAGCC CGACGCGCGA ACCGGGATTA GTCCCGCGCG CGCACACGTGGCGGCCGCCG 12840 ACCTGGTAAC CGCATACGAG CAGACGGTGA ACCAGGAGAT TAACTTTCAAAAAAGCTTTA 12900 ACAACCACGT GCGTACGCTT GTGGCGCGCG AGGAGGTGGC TATAGGACTGATGCATCTGT 12960 GGGACTTTGT AAGCGCGCTG GAGCAAAACC CAAATAGCAA GCCGCTCATGGCGCAGCTGT 13020 TCCTTATAGT GCAGCACAGC AGGGACAACG AGGCATTCAG GGATGCGCTGCTAAACATAG 13080 TAGAGCCCGA GGGCCGCTGG CTGCTCGATT TGATAAACAT CCTGCAGAGCATAGTGGTGC 13140 AGGAGCGCAG CTTGAGCCTG GCTGACAAGG TGGCCGCCAT CAACTATTCCATGCTTAGCC 13200 TGGGCAAGTT TTACGCCCGC AAGATATACC ATACCCCTTA CGTTCCCATAGACAAGGAGG 13260 TAAAGATCGA GGGGTTCTAC ATGCGCATGG CGCTGAAGGT GCTTACCTTGAGCGACGACC 13320 TGGGCGTTTA TCGCAACGAG CGCATCCACA AGGCCGTGAG CGTGAGCCGGCGGCGCGAGC 13380 TCAGCGACCG CGAGCTGATG CACAGCCTGC AAAGGGCCCT GGCTGGCACGGGCAGCGGCG 13440 ATAGAGAGGC CGAGTCCTAC TTTGACGCGG GCGCTGACCT GCGCTGGGCCCCAAGCCGAC 13500 GCGCCCTGGA GGCAGCTGGG GCCGGACCTG GGCTGGCGGT GGCACCCGCGCGCGCTGGCA 13560 ACGTCGGCGG CGTGGAGGAA TATGACGAGG ACGATGAGTA CGAGCCAGAGGACGGCGAGT 13620 ACTAAGCGGT GATGTTTCTG ATCAGATGAT GCAAGACGCA ACGGACCCGGCGGTGCGGGC 13680 GGCGCTGCAG AGCCAGCCGT CCGGCCTTAA CTCCACGGAC GACTGGCGCCAGGTCATGGA 13740 CCGCATCATG TCGCTGACTG CGCGCAATCC TGACGCGTTC CGGCAGCAGCCGCAGGCCAA 13800 CCGGCTCTCC GCAATTCTGG AAGCGGTGGT CCCGGCGCGC GCAAACCCCACGCACGAGAA 13860 GGTGCTGGCG ATCGTAAACG CGCTGGCCGA AAACAGGGCC ATCCGGCCCGACGAGGCCGG 13920 CCTGGTCTAC GACGCGCTGC TTCAGCGCGT GGCTCGTTAC AACAGCGGCAACGTGCAGAC 13980 CAACCTGGAC CGGCTGGTGG GGGATGTGCG CGAGGCCGTG GCGCAGCGTGAGCGCGCGCA 14040 GCAGCAGGGC AACCTGGGCT CCATGGTTGC ACTAAACGCC TTCCTGAGTACACAGCCCGC 14100 CAACGTGCCG CGGGGACAGG AGGACTACAC CAACTTTGTG AGCGCACTGCGGCTAATGGT 14160 GACTGAGACA CCGCAAAGTG AGGTGTACCA GTCTGGGCCA GACTATTTTTTCCAGACCAG 14220 TAGACAAGGC CTGCAGACCG TAAACCTGAG CCAGGCTTTC AAAAACTTGCAGGGGCTGTG 14280 GGGGGTGCGG GCTCCCACAG GCGACCGCGC GACCGTGTCT AGCTTGCTGACGCCCAACTC 14340 GCGCCTGTTG CTGCTGCTAA TAGCGCCCTT CACGGACAGT GGCAGCGTGTCCCGGGACAC 14400 ATACCTAGGT CACTTGCTGA CACTGTACCG CGAGGCCATA GGTCAGGCGCATGTGGACGA 14460 GCATACTTTC CAGGAGATTA CAAGTGTCAG CCGCGCGCTG GGGCAGGAGGACACGGGCAG 14520 CCTGGAGGCA ACCCTAAACT ACCTGCTGAC CAACCGGCGG CAGAAGATCCCCTCGTTGCA 14580 CAGTTTAAAC AGCGAGGAGG AGCGCATTTT GCGCTACGTG CAGCAGAGCGTGAGCCTTAA 14640 CCTGATGCGC GACGGGGTAA CGCCCAGCGT GGCGCTGGAC ATGACCGCGCGCAACATGGA 14700 ACCGGGCATG TATGCCTCAA ACCGGCCGTT TATCAACCGC CTAATGGACTACTTGCATCG 14760 CGCGGCCGCC GTGAACCCCG AGTATTTCAC CAATGCCATC TTGAACCCGCACTGGCTACC 14820 GCCCCCTGGT TTCTACACCG GGGGATTCGA GGTGCCCGAG GGTAACGATGGATTCCTCTG 14880 GGACGACATA GACGACAGCG TGTTTTCCCC GCAACCGCAG ACCCTGCTAGAGTTGCAACA 14940 GCGCGAGCAG GCAGAGGCGG CGCTGCGAAA GGAAAGCTTC CGCAGGCCAAGCAGCTTGTC 15000 CGATCTAGGC GCTGCGGCCC CGCGGTCAGA TGCTAGTAGC CCATTTCCAAGCTTGATAGG 15060 GTCTCTTACC AGCACTCGCA CCACCCGCCC GCGCCTGCTG GGCGAGGAGGAGTACCTAAA 15120 CAACTCGCTG CTGCAGCCGC AGCGCGAAAA AAACCTGCCT CCGGCATTTCCCAACAACGG 15180 GATAGAGAGC CTAGTGGACA AGATGAGTAG ATGGAAGACG TACGCGCAGGAGCACAGGGA 15240 CGTGCCAGGC CCGCGCCCGC CCACCCGTCG TCAAAGGCAC GACCGTCAGCGGGGTCTGGT 15300 GTGGGAGGAC GATGACTCGG CAGACGACAG CAGCGTCCTG GATTTGGGAGGGAGTGGCAA 15360 CCCGTTTGCG CACCTTCGCC CCAGGCTGGG GAGAATGTTT TAAAAAAAAAAAAGCATGAT 15420 GCAAAATAAA AAACTCACCA AGGCCATGGC ACCGAGCGTT GGTTTTCTTGTATTCCCCTT 15480 AGTATGCGGC GCGCGGCGAT GTATGAGGAA GGTCCTCCTC CCTCCTACGAGAGTGTGGTG 15540 AGCGCGGCGC CAGTGGCGGC GGCGCTGGGT TCTCCCTTCG ATGCTCCCCTGGACCCGCCG 15600 TTTGTGCCTC CGCGGTACCT GCGGCCTACC GGGGGGAGAA ACAGCATCCGTTACTCTGAG 15660 TTGGCACCCC TATTCGACAC CACCCGTGTG TACCTGGTGG ACAACAAGTCAACGGATGTG 15720 GCATCCCTGA ACTACCAGAA CGACCACAGC AACTTTCTGA CCACGGTCATTCAAAACAAT 15780 GACTACAGCC CGGGGGAGGC AAGCACACAG ACCATCAATC TTGACGACCGGTCGCACTGG 15840 GGCGGCGACC TGAAAACCAT CCTGCATACC AACATGCCAA ATGTGAACGAGTTCATGTTT 15900 ACCAATAAGT TTAAGGCGCG GGTGATGGTG TCGCGCTTGC CTACTAAGGACAATCAGGTG 15960 GAGCTGAAAT ACGAGTGGGT GGAGTTCACG CTGCCCGAGG GCAACTACTCCGAGACCATG 16020 ACCATAGACC TTATGAACAA CGCGATCGTG GAGCACTACT TGAAAGTGGGCAGACAGAAC 16080 GGGGTTCTGG AAAGCGACAT CGGGGTAAAG TTTGACACCC GCAACTTCAGACTGGGGTTT 16140 GACCCCGTCA CTGGTCTTGT CATGCCTGGG GTATATACAA ACGAAGCCTTCCATCCAGAC 16200 ATCATTTTGC TGCCAGGATG CGGGGTGGAC TTCACCCACA GCCGCCTGAGCAACTTGTTG 16260 GGCATCCGCA AGCGGCAACC CTTCCAGGAG GGCTTTAGGA TCACCTACGATGATCTGGAG 16320 GGTGGTAACA TTCCCGCACT GTTGGATGTG GACGCCTACC AGGCGAGCTTGAAAGATGAC 16380 ACCGAACAGG GCGGGGGTGG CGCAGGCGGC AGCAACAGCA GTGGCAGCGGCGCGGAAGAG 16440 AACTCCAACG CGGCAGCCGC GGCAATGCAG CCGGTGGAGG ACATGAACGATCATGCCATT 16500 CGCGGCGACA CCTTTGCCAC ACGGGCTGAG GAGAAGCGCG CTGAGGCCGAAGCAGCGGCC 16560 GAAGCTGCCG CCCCCGCTGC GCAACCCGAG GTCGAGAAGC CTCAGAAGAAACCGGTGATC 16620 AAACCCCTGA CAGAGGACAG CAAGAAACGC AGTTACAACC TAATAAGCAATGACAGCACC 16680 TTCACCCAGT ACCGCAGCTG GTACCTTGCA TACAACTACG GCGACCCTCAGACCGGAATC 16740 CGCTCATGGA CCCTGCTTTG CACTCCTGAC GTAACCTGCG GCTCGGAGCAGGTCTACTGG 16800 TCGTTGCCAG ACATGATGCA AGACCCCGTG ACCTTCCGCT CCACGCGCCAGATCAGCAAC 16860 TTTCCGGTGG TGGGCGCCGA GCTGTTGCCC GTGCACTCCA AGAGCTTCTACAACGACCAG 16920 GCCGTCTACT CCCAACTCAT CCGCCAGTTT ACCTCTCTGA CCCACGTGTTCAATCGCTTT 16980 CCCGAGAACC AGATTTTGGC GCGCCCGCCA GCCCCCACCA TCACCACCGTCAGTGAAAAC 17040 GTTCCTGCTC TCACAGATCA CGGGACGCTA CCGCTGCGCA ACAGCATCGGAGGAGTCCAG 17100 CGAGTGACCA TTACTGACGC CAGACGCCGC ACCTGCCCCT ACGTTTACAAGGCCCTGGGC 17160 ATAGTCTCGC CGCGCGTCCT ATCGAGCCGC ACTTTTTGAG CAAGCATGTCCATCCTTATA 17220 TCGCCCAGCA ATAACACAGG CTGGGGCCTG CGCTTCCCAA GCAAGATGTTTGGCGGGGCC 17280 AAGAAGCGCT CCGACCAACA CCCAGTGCGC GTGCGCGGGC ACTACCGCGCGCCCTGGGGC 17340 GCGCACAAAC GCGGCCGCAC TGGGCGCACC ACCGTCGATG ACGCCATCGACGCGGTGGTG 17400 GAGGAGGCGC GCAACTACAC GCCCACGCCG CCACCAGTGT CCACAGTGGACGCGGCCATT 17460 CAGACCGTGG TGCGCGGAGC CCGGCGCTAT GCTAAAATGA AGAGACGGCGGAGGCGCGTA 17520 GCACGTCGCC ACCGCCGCCG ACCCGGCACT GCCGCCCAAC GCGCGGCGGCGGCCCTGCTT 17580 AACCGCGCAC GTCGCACCGG CCGACGGGCG GCCATGCGGG CCGCTCGAAGGCTGGCCGCG 17640 GGTATTGTCA CTGTGCCCCC CAGGTCCAGG CGACGAGCGG CCGCCGCAGCAGCCGCGGCC 17700 ATTAGTGCTA TGACTCAGGG TCGCAGGGGC AACGTGTATT GGGTGCGCGACTCGGTTAGC 17760 GGCCTGCGCG TGCCCGTGCG CACCCGCCCC CCGCGCAACT AGATTGCAAGAAAAAACTAC 17820 TTAGACTCGT ACTGTTGTAT GTATCCAGCG GCGGCGGCGC GCAACGAAGCTATGTCCAAG 17880 CGCAAAATCA AAGAAGAGAT GCTCCAGGTC ATCGCGCCGG AGATCTATGGCCCCCCGAAG 17940 AAGGAAGAGC AGGATTACAA GCCCCGAAAG CTAAAGCGGG TCAAAAAGAAAAAGAAAGAT 18000 GATGATGATG AACTTGACGA CGAGGTGGAA CTGCTGCACG CTACCGCGCCCAGGCGACGG 18060 GTACAGTGGA AAGGTCGACG CGTAAAACGT GTTTTGCGAC CCGGCACCACCGTAGTCTTT 18120 ACGCCCGGTG AGCGCTCCAC CCGCACCTAC AAGCGCGTGT ATGATGAGGTGTACGGCGAC 18180 GAGGACCTGC TTGAGCAGGC CAACGAGCGC CTCGGGGAGT TTGCCTACGGAAAGCGGCAT 18240 AAGGACATGC TGGCGTTGCC GCTGGACGAG GGCAACCCAA CACCTAGCCTAAAGCCCGTA 18300 ACACTGCAGC AGGTGCTGCC CGCGCTTGCA CCGTCCGAAG AAAAGCGCGGCCTAAAGCGC 18360 GAGTCTGGTG ACTTGGCACC CACCGTGCAG CTGATGGTAC CCAAGCGCCAGCGACTGGAA 18420 GATGTCTTGG AAAAAATGAC CGTGGAACCT GGGCTGGAGC CCGAGGTCCGCGTGCGGCCA 18480 ATCAAGCAGG TGGCGCCGGG ACTGGGCGTG CAGACCGTGG ACGTTCAGATACCCACTACC 18540 AGTAGCACCA GTATTGCCAC CGCCACAGAG GGCATGGAGA CACAAACGTCCCCGGTTGCC 18600 TCAGCGGTGG CGGATGCCGC GGTGCAGGCG GTCGCTGCGG CCGCGTCCAAGACCTCTACG 18660 GAGGTGCAAA CGGACCCGTG GATGTTTCGC GTTTCAGCCC CCCGGCGCCCGCGCGGTTCG 18720 AGGAAGTACG GCGCCGCCAG CGCGCTACTG CCCGAATATG CCCTACATCCTTCCATTGCG 18780 CCTACCCCCG GCTATCGTGG CTACACCTAC CGCCCCAGAA GACGAGCAACTACCCGACGC 18840 CGAACCACCA CTGGAACCCG CCGCCGCCGT CGCCGTCGCC AGCCCGTGCTGGCCCCGATT 18900 TCCGTGCGCA GGGTGGCTCG CGAAGGAGGC AGGACCCTGG TGCTGCCAACAGCGCGCTAC 18960 CACCCCAGCA TCGTTTAAAA GCCGGTCTTT GTGGTTCTTG CAGATATGGCCCTCACCTGC 19020 CGCCTCCGTT TCCCGGTGCC GGGATTCCGA GGAAGAATGC ACCGTAGGAGGGGCATGGCC 19080 GGCCACGGCC TGACGGGCGG CATGCGTCGT GCGCACCACC GGCGGCGGCGCGCGTCGCAC 19140 CGTCGCATGC GCGGCGGTAT CCTGCCCCTC CTTATTCCAC TGATCGCCGCGGCGATTGGC 19200 GCCGTGCCCG GAATTGCATC CGTGGCCTTG CAGGCGCAGA GACACTGATTAAAAACAAGT 19260 TGCATGTGGA AAAATCAAAA TAAAAAGTCT GGACTCTCAC GCTCGCTTGGTCCTGTAACT 19320 ATTTTGTAGA ATGGAAGACA TCAACTTTGC GTCTCTGGCC CCGCGACACGGCTCGCGCCC 19380 GTTCATGGGA AACTGGCAAG ATATCGGCAC CAGCAATATG AGCGGTGGCGCCTTCAGCTG 19440 GGGCTCGCTG TGGAGCGGCA TTAAAAATTT CGGTTCCACC GTTAAGAACTATGGCAGCAA 19500 GGCCTGGAAC AGCAGCACAG GCCAGATGCT GAGGGATAAG TTGAAAGAGCAAAATTTCCA 19560 ACAAAAGGTG GTAGATGGCC TGGCCTCTGG CATTAGCGGG GTGGTGGACCTGGCCAACCA 19620 GGCAGTGCAA AATAAGATTA ACAGTAAGCT TGATCCCCGC CCTCCCGTAGAGGAGCCTCC 19680 ACCGGCCGTG GAGACAGTGT CTCCAGAGGG GCGTGGCGAA AAGCGTCCGCGCCCCGACAG 19740 GGAAGAAACT CTGGTGACGC AAATAGACGA GCCTCCCTCG TACGAGGAGGCACTAAAGCA 19800 AGGCCTGCCC ACCACCCGTC CCATCGCGCC CATGGCTACC GGAGTGCTGGGCCAGCACAC 19860 ACCCGTAACG CTGGACCTGC CTCCCCCCGC CGACACCCAG CAGAAACCTGTGCTGCCAGG 19920 CCCGACCGCC GTTGTTGTAA CCCGTCCTAG CCGCGCGTCC CTGCGCCGCGCCGCCAGCGG 19980 TCCGCGATCG TTGCGGCCCG TAGCCAGTGG CAACTGGCAA AGCACACTGAACAGCATCGT 20040 GGGTCTGGGG GTGCAATCCC TGAAGCGCCG ACGATGCTTC TGAATAGCTAACGTGTCGTA 20100 TGTGTGTCAT GTATGCGTCC ATGTCGCCGC CAGAGGAGCT GCTGAGCCGCCGCGCGCCCG 20160 CTTTCCAAGA TGGCTACCCC TTCGATGATG CCGCAGTGGT CTTACATGCACATCTCGGGC 20220 CAGGACGCCT CGGAGTACCT GAGCCCCGGG CTGGTGCAGT TTGCCCGCGCCACCGAGACG 20280 TACTTCAGCC TGAATAACAA GTTTAGAAAC CCCACGGTGG CGCCTACGCACGACGTGACC 20340 ACAGACCGGT CCCAGCGTTT GACGCTGCGG TTCATCCCTG TGGACCGTGAGGATACTGCG 20400 TACTCGTACA AGGCGCGGTT CACCCTAGCT GTGGGTGATA ACCGTGTGCTGGACATGGCT 20460 TCCACGTACT TTGACATCCG CGGCGTGCTG GACAGGGGCC CTACTTTTAAGCCCTACTCT 20520 GGCACTGCCT ACAACGCCCT GGCTCCCAAG GGTGCCCCAA ATCCTTGCGAATGGGATGAA 20580 GCTGCTACTG CTCTTGAAAT AAACCTAGAA GAAGAGGACG ATGACAACGAAGACGAAGTA 20640 GACGAGCAAG CTGAGCAGCA AAAAACTCAC GTATTTGGGC AGGCGCCTTATTCTGGTATA 20700 AATATTACAA AGGAGGGTAT TCAAATAGGT GTCGAAGGTC AAACACCTAAATATGCCGAT 20760 AAAACATTTC AACCTGAACC TCAAATAGGA GAATCTCAGT GGTACGAAACTGAAATTAAT 20820 CATGCAGCTG GGAGAGTCCT TAAAAAGACT ACCCCAATGA AACCATGTTACGGTTCATAT 20880 GCAAAACCCA CAAATGAAAA TGGAGGGCAA GGCATTCTTG TAAAGCAACAAAATGGAAAG 20940 CTAGAAAGTC AAGTGGAAAT GCAATTTTTC TCAACTACTG AGGCGACCGCAGGCAATGGT 21000 GATAACTTGA CTCCTAAAGT GGTATTGTAC AGTGAAGATG TAGATATAGAAACCCCAGAC 21060 ACTCATATTT CTTACATGCC CACTATTAAG GAAGGTAACT CACGAGAACTAATGGGCCAA 21120 CAATCTATGC CCAACAGGCC TAATTACATT GCTTTTAGGG ACAATTTTATTGGTCTAATG 21180 TATTACAACA GCACGGGTAA TATGGGTGTT CTGGCGGGCC AAGCATCGCAGTTGAATGCT 21240 GTTGTAGATT TGCAAGACAG AAACACAGAG CTTTCATACC AGCTTTTGCTTGATTCCATT 21300 GGTGATAGAA CCAGGTACTT TTCTATGTGG AATCAGGCTG TTGACAGCTATGATCCAGAT 21360 GTTAGAATTA TTGAAAATCA TGGAACTGAA GATGAACTTC CAAATTACTGCTTTCCACTG 21420 GGAGGTGTGA TTAATACAGA GACTCTTACC AAGGTAAAAC CTAAAACAGGTCAGGAAAAT 21480 GGATGGGAAA AAGATGCTAC AGAATTTTCA GATAAAAATG AAATAAGAGTTGGAAATAAT 21540 TTTGCCATGG AAATCAATCT AAATGCCAAC CTGTGGAGAA ATTTCCTGTACTCCAACATA 21600 GCGCTGTATT TGCCCGACAA GCTAAAGTAC AGTCCTTCCA ACGTAAAAATTTCTGATAAC 21660 CCAAACACCT ACGACTACAT GAACAAGCGA GTGGTGGCTC CCGGGTTAGTGGACTGCTAC 21720 ATTAACCTTG GAGCACGCTG GTCCCTTGAC TATATGGACA ACGTCAACCCATTTAACCAC 21780 CACCGCAATG CTGGCCTGCG CTACCGCTCA ATGTTGCTGG GCAATGGTCGCTATGTGCCC 21840 TTCCACATCC AGGTGCCTCA GAAGTTCTTT GCCATTAAAA ACCTCCTTCTCCTGCCGGGC 21900 TCATACACCT ACGAGTGGAA CTTCAGGAAG GATGTTAACA TGGTTCTGCAGAGCTCCCTA 21960 GGAAATGACC TAAGGGTTGA CGGAGCCAGC ATTAAGTTTG ATAGCATTTGCCTTTACGCC 22020 ACCTTCTTCC CCATGGCCCA CAACACCGCC TCCACGCTTG AGGCCATGCTTAGAAACGAC 22080 ACCAACGACC AGTCCTTTAA CGACTATCTC TCCGCCGCCA ACATGCTCTACCCTATACCC 22140 GCCAACGCTA CCAACGTGCC CATATCCATC CCCTCCCGCA ACTGGGCGGCTTTCCGCGGC 22200 TGGGCCTTCA CGCGCCTTAA GACTAAGGAA ACCCCATCAC TGGGCTCGGGCTACGACCCT 22260 TATTACACCT ACTCTGGCTC TATACCCTAC CTAGATGGAA CCTTTTACCTCAACCACACC 22320 TTTAAGAAGG TGGCCATTAC CTTTGACTCT TCTGTCAGCT GGCCTGGCAATGACCGCCTG 22380 CTTACCCCCA ACGAGTTTGA AATTAAGCGC TCAGTTGACG GGGAGGGTTACAACGTTGCC 22440 CAGTGTAACA TGACCAAAGA CTGGTTCCTG GTACAAATGC TAGCTAACTACAACATTGGC 22500 TACCAGGGCT TCTATATCCC AGAGAGCTAC AAGGACCGCA TGTACTCCTTCTTTAGAAAC 22560 TTCCAGCCCA TGAGCCGTCA GGTGGTGGAT GATACTAAAT ACAAGGACTACCAACAGGTG 22620 GGCATCCTAC ACCAACACAA CAACTCTGGA TTTGTTGGCT ACCTTGCCCCCACCATGCGC 22680 GAAGGACAGG CCTACCCTGC TAACTTCCCC TATCCGCTTA TAGGCAAGACCGCAGTTGAC 22740 AGCATTACCC AGAAAAAGTT TCTTTGCGAT CGCACCCTTT GGCGCATCCCATTCTCCAGT 22800 AACTTTATGT CCATGGGCGC ACTCACAGAC CTGGGCCAAA ACCTTCTCTACGCCAACTCC 22860 GCCCACGCGC TAGACATGAC TTTTGAGGTG GATCCCATGG ACGAGCCCACCCTTCTTTAT 22920 GTTTTGTTTG AAGTCTTTGA CGTGGTCCGT GTGCACCGGC CGCACCGCGGCGTCATCGAA 22980 ACCGTGTACC TGCGCACGCC CTTCTCGGCC GGCAACGCCA CAACATAAAGAAGCAAGCAA 23040 CATCAACAAC AGCTGCCGCC ATGGGCTCCA GTGAGCAGGA ACTGAAAGCCATTGTCAAAG 23100 ATCTTGGTTG TGGGCCATAT TTTTTGGGCA CCTATGACAA GCGCTTTCCAGGCTTTGTTT 23160 CTCCACACAA GCTCGCCTGC GCCATAGTCA ATACGGCCGG TCGCGAGACTGGGGGCGTAC 23220 ACTGGATGGC CTTTGCCTGG AACCCGCACT CAAAAACATG CTACCTCTTTGAGCCCTTTG 23280 GCTTTTCTGA CCAGCGACTC AAGCAGGTTT ACCAGTTTGA GTACGAGTCACTCCTGCGCC 23340 GTAGCGCCAT TGCTTCTTCC CCCGACCGCT GTATAACGCT GGAAAAGTCCACCCAAAGCG 23400 TACAGGGGCC CAACTCGGCC GCCTGTGGAC TATTCTGCTG CATGTTTCTCCACGCCTTTG 23460 CCAACTGGCC CCAAACTCCC ATGGATCACA ACCCCACCAT GAACCTTATTACCGGGGTAC 23520 CCAACTCCAT GCTCAACAGT CCCCAGGTAC AGCCCACCCT GCGTCGCAACCAGGAACAGC 23580 TCTACAGCTT CCTGGAGCGC CACTCGCCCT ACTTCCGCAG CCACAGTGCGCAGATTAGGA 23640 GCGCCACTTC TTTTTGTCAC TTGAAAAACA TGTAAAAATA ATGTACTAGAGACACTTTCA 23700 ATAAAGGCAA ATGCTTTTAT TTGTACACTC TCGGGTGATT ATTTACCCCCACCCTTGCCG 23760 TCTGCGCCGT TTAAAAATCA AAGGGGTTCT GCCGCGCATC GCTATGCGCCACTGGCAGGG 23820 ACACGTTGCG ATACTGGTGT TTAGTGCTCC ACTTAAACTC AGGCACAACCATCCGCGGCA 23880 GCTCGGTGAA GTTTTCACTC CACAGGCTGC GCACCATCAC CAACGCGTTTAGCAGGTCGG 23940 GCGCCGATAT CTTGAAGTCG CAGTTGGGGC CTCCGCCCTG CGCGCGCGAGTTGCGATACA 24000 CAGGGTTGCA GCACTGGAAC ACTATCAGCG CCGGGTGGTG CACGCTGGCCAGCACGCTCT 24060 TGTCGGAGAT CAGATCCGCG TCCAGGTCCT CCGCGTTGCT CAGGGCGAACGGAGTCAACT 24120 TTGGTAGCTG CCTTCCCAAA AAGGGCGCGT GCCCAGGCTT TGAGTTGCACTCGCACCGTA 24180 GTGGCATCAA AAGGTGACCG TGCCCGGTCT GGGCGTTAGG ATACAGCGCCTGCATAAAAG 24240 CCTTGATCTG CTTAAAAGCC ACCTGAGCCT TTGCGCCTTC AGAGAAGAACATGCCGCAAG 24300 ACTTGCCGGA AAACTGATTG GCCGGACAGG CCGCGTCGTG CACGCAGCACCTTGCGTCGG 24360 TGTTGGAGAT CTGCACCACA TTTCGGCCCC ACCGGTTCTT CACGATCTTGGCCTTGCTAG 24420 ACTGCTCCTT CAGCGCGCGC TGCCCGTTTT CGCTCGTCAC ATCCATTTCAATCACGTGCT 24480 CCTTATTTAT CATAATGCTT CCGTGTAGAC ACTTAAGCTC GCCTTCGATCTCAGCGCAGC 24540 GGTGCAGCCA CAACGCGCAG CCCGTGGGCT CGTGATGCTT GTAGGTCACCTCTGCAAACG 24600 ACTGCAGGTA CGCCTGCAGG AATCGCCCCA TCATCGTCAC AAAGGTCTTGTTGCTGGTGA 24660 AGGTCAGCTG CAACCCGCGG TGCTCCTCGT TCAGCCAGGT CTTGCATACGGCCGCCAGAG 24720 CTTCCACTTG GTCAGGCAGT AGTTTGAAGT TCGCCTTTAG ATCGTTATCCACGTGGTACT 24780 TGTCCATCAG CGCGCGCGCA GCCTCCATGC CCTTCTCCCA CGCAGACACGATCGGCACAC 24840 TCAGCGGGTT CATCACCGTA ATTTCACTTT CCGCTTCGCT GGGCTCTTCCTCTTCCTCTT 24900 GCGTCCGCAT ACCACGCGCC ACTGGGTCGT CTTCATTCAG CCGCCGCACTGTGCGCTTAC 24960 CTCCTTTGCC ATGCTTGATT AGCACCGGTG GGTTGCTGAA ACCCACCATTTGTAGCGCCA 25020 CATCTTCTCT TTCTTCCTCG CTGTCCACGA TTACCTCTGG TGATGGCGGGCGCTCGGGCT 25080 TGGGAGAAGG GCGCTTCTTT TTCTTCTTGG GCGCAATGGC CAAATCCGCCGCCGAGGTCG 25140 ATGGCCGCGG GCTGGGTGTG CGCGGCACCA GCGCGTCTTG TGATGAGTCTTCCTCGTCCT 25200 CGGACTCGAT ACGCCGCCTC ATCCGCTTTT TTGGGGGCGC CCGGGGAGGCGGCGGCGACG 25260 GGGACGGGGA CGACACGTCC TCCATGGTTG GGGGACGTCG CGCCGCACCGCGTCCGCGCT 25320 CGGGGGTGGT TTCGCGCTGC TCCTCTTCCC GACTGGCCAT TTCCTTCTCCTATAGGCAGA 25380 AAAAGATCAT GGAGTCAGTC GAGAAGAAGG ACAGCCTAAC CGCCCCCTCTGAGTTCGCCA 25440 CCACCGCCTC CACCGATGCC GCCAACGCGC CTACCACCTT CCCCGTCGAGGCACCCCCGC 25500 TTGAGGAGGA GGAAGTGATT ATCGAGCAGG ACCCAGGTTT TGTAAGCGAAGACGACGAGG 25560 ACCGCTCAGT ACCAACAGAG GATAAAAAGC AAGACCAGGA CAACGCAGAGGCAAACGAGG 25620 AACAAGTCGG GCGGGGGGAC GAAAGGCATG GCGACTACCT AGATGTGGGAGACGACGTGC 25680 TGTTGAAGCA TCTGCAGCGC CAGTGCGCCA TTATCTGCGA CGCGTTGCAAGAGCGCAGCG 25740 ATGTGCCCCT CGCCATAGCG GATGTCAGCC TTGCCTACGA ACGCCACCTATTCTCACCGC 25800 GCGTACCCCC CAAACGCCAA GAAAACGGCA CATGCGAGCC CAACCCGCGCCTCAACTTCT 25860 ACCCCGTATT TGCCGTGCCA GAGGTGCTTG CCACCTATCA CATCTTTTTCCAAAACTGCA 25920 AGATACCCCT ATCCTGCCGT GCCAACCGCA GCCGAGCGGA CAAGCAGCTGGCCTTGCGGC 25980 AGGGCGCTGT CATACCTGAT ATCGCCTCGC TCAACGAAGT GCCAAAAATCTTTGAGGGTC 26040 TTGGACGCGA CGAGAAGCGC GCGGCAAACG CTCTGCAACA GGAAAACAGCGAAAATGAAA 26100 GTCACTCTGG AGTGTTGGTG GAACTCGAGG GTGACAACGC GCGCCTAGCCGTACTAAAAC 26160 GCAGCATCGA GGTCACCCAC TTTGCCTACC CGGCACTTAA CCTACCCCCCAAGGTCATGA 26220 GCACAGTCAT GAGTGAGCTG ATCGTGCGCC GTGCGCAGCC CCTGGAGAGGGATGCAAATT 26280 TGCAAGAACA AACAGAGGAG GGCCTACCCG CAGTTGGCGA CGAGCAGCTAGCGCGCTGGC 26340 TTCAAACGCG CGAGCCTGCC GACTTGGAGG AGCGACGCAA ACTAATGATGGCCGCAGTGC 26400 TCGTTACCGT GGAGCTTGAG TGCATGCAGC GGTTCTTTGC TGACCCGGAGATGCAGCGCA 26460 AGCTAGAGGA AACATTGCAC TACACCTTTC GACAGGGCTA CGTACGCCAGGCCTGCAAGA 26520 TCTCCAACGT GGAGCTCTGC AACCTGGTCT CCTACCTTGG AATTTTGCACGAAAACCGCC 26580 TTGGGCAAAA CGTGCTTCAT TCCACGCTCA AGGGCGAGGC GCGCCGCGACTACGTCCGCG 26640 ACTGCGTTTA CTTATTTCTA TGCTACACCT GGCAGACGGC CATGGGCGTTTGGCAGCAGT 26700 GCTTGGAGGA GTGCAACCTC AAGGAGCTGC AGAAACTGCT AAAGCAAAACTTGAAGGACC 26760 TATGGACGGC CTTCAACGAG CGCTCCGTGG CCGCGCACCT GGCGGACATCATTTTCCCCG 26820 AACGCCTGCT TAAAACCCTG CAACAGGGTC TGCCAGACTT CACCAGTCAAAGCATGTTGC 26880 AGAACTTTAG GAACTTTATC CTAGAGCGCT CAGGAATCTT GCCCGCCACCTGCTGTGCAC 26940 TTCCTAGCGA CTTTGTGCCC ATTAAGTACC GCGAATGCCC TCCGCCGCTTTGGGGCCACT 27000 GCTACCTTCT GCAGCTAGCC AACTACCTTG CCTACCACTC TGACATAATGGAAGACGTGA 27060 GCGGTGACGG TCTACTGGAG TGTCACTGTC GCTGCAACCT ATGCACCCCGCACCGCTCCC 27120 TGGTTTGCAA TTCGCAGCTG CTTAACGAAA GTCAAATTAT CGGTACCTTTGAGCTGCAGG 27180 GTCCCTCGCC TGACGAAAAG TCCGCGGCTC CGGGGTTGAA ACTCACTCCGGGGCTGTGGA 27240 CGTCGGCTTA CCTTCGCAAA TTTGTACCTG AGGACTACCA CGCCCACGAGATTAGGTTCT 27300 ACGAAGACCA ATCCCGCCCG CCAAATGCGG AGCTTACCGC CTGCGTCATTACCCAGGGCC 27360 ACATTCTTGG CCAATTGCAA GCCATCAACA AAGCCCGCCA AGAGTTTCTGCTACGAAAGG 27420 GACGGGGGGT TTACTTGGAC CCCCAGTCCG GCGAGGAGCT CAACCCAATCCCCCCGCCGC 27480 CGCAGCCCTA TCAGCAGCAG CCGCGGGCCC TTGCTTCCCA GGATGGCACCCAAAAAGAAG 27540 CTGCAGCTGC CGCCGCCACC CACGGACGAG GAGGAATACT GGGACAGTCAGGCAGAGGAG 27600 GTTTTGGACG AGGAGGAGGA GGACATGATG GAAGACTGGG AGAGCCTAGACGAGGAAGCT 27660 TCCGAGGTCG AAGAGGTGTC AGACGAAACA CCGTCACCCT CGGTCGCATTCCCCTCGCCG 27720 GCGCCCCAGA AATCGGCAAC CGGTTCCAGC ATGGCTACAA CCTCCGCTCCTCAGGCGCCG 27780 CCGGCACTGC CCGTTCGCCG ACCCAACCGT AGATGGGACA CCACTGGAACCAGGGCCGGT 27840 AAGTCCAAGC AGCCGCCGCC GTTAGCCCAA GAGCAACAAC AGCGCCAAGGCTACCGCTCA 27900 TGGCGCGGGC ACAAGAACGC CATAGTTGCT TGCTTGCAAG ACTGTGGGGGCAACATCTCC 27960 TTCGCCCGCC GCTTTCTTCT CTACCATCAC GGCGTGGCCT TCCCCCGTAACATCCTGCAT 28020 TACTACCGTC ATCTCTACAG CCCATACTGC ACCGGCGGCA GCGGCAGCGGCAGCAACAGC 28080 AGCGGCCACA CAGAAGCAAA GGCGACCGGA TAGCAAGACT CTGACAAAGCCCAAGAAATC 28140 CACAGCGGCG GCAGCAGCAG GAGGAGGAGC GCTGCGTCTG GCGCCCAACGAACCCGTATC 28200 GACCCGCGAG CTTAGAAACA GGATTTTTCC CACTCTGTAT GCTATATTTCAACAGAGCAG 28260 GGGCCAAGAA CAAGAGCTGA AAATAAAAAA CAGGTCTCTG CGATCCCTCACCCGCAGCTG 28320 CCTGTATCAC AAAAGCGAAG ATCAGCTTCG GCGCACGCTG GAAGACGCGGAGGCTCTCTT 28380 CAGTAAATAC TGCGCGCTGA CTCTTAAGGA CTAGTTTCGC GCCCTTTCTCAAATTTAAGC 28440 GCGAAAACTA CGTCATCTCC AGCGGCCACA CCCGGCGCCA GCACCTGTCGTCAGCGCCAT 28500 TATGAGCAAG GAAATTCCCA CGCCCTACAT GTGGAGTTAC CAGCCACAAATGGGACTTGC 28560 GGCTGGAGCT GCCCAAGACT ACTCAACCCG AATAAACTAC ATGAGCGCGGGACCCCACAT 28620 GATATCCCGG GTCAACGGAA TCCGCGCCCA CCGAAACCGA ATTCTCTTGGAACAGGCGGC 28680 TATTACCACC ACACCTCGTA ATAACCTTAA TCCCCGTAGT TGGCCCGCTGCCCTGGTGTA 28740 CCAGGAAAGT CCCGCTCCCA CCACTGTGGT ACTTCCCAGA GACGCCCAGGCCGAAGTTCA 28800 GATGACTAAC TCAGGGGCGC AGCTTGCGGG CGGCTTTCGT CACAGGGTGCGGTCGCCCGG 28860 GCAGGGTATA ACTCACCTGA CAATCAGAGG GCGAGGTATT CAGCTCAACGACGAGTCGGT 28920 GAGCTCCTCG CTTGGTCTCC GTCCGGACGG GACATTTCAG ATCGGCGGCGCCGGCCGTCC 28980 TTCATTCACG CCTCGTCAGG CAATCCTAAC TCTGCAGACC TCGTCCTCTGAGCCGCGCTC 29040 TGGAGGCATT GGAACTCTGC AATTTATTGA GGAGTTTGTG CCATCGGTCTACTTTAACCC 29100 CTTCTCGGGA CCTCCCGGCC ACTATCCGGA TCAATTTATT CCTAACTTTGACGCGGTAAA 29160 GGACTCGGCG GACGGCTACG ACTGAATGTT AAGTGGAGAG GCAGAGCAACTGCGCCTGAA 29220 ACACCTGGTC CACTGTCGCC GCCACAAGTG CTTTGCCCGC GACTCCGGTGAGTTTTGCTA 29280 CTTTGAATTG CCCGAGGATC ATATCGAGGG CCCGGCGCAC GGCGTCCGGCTTACCGCCCA 29340 GGGAGAGCTT GCCCGTAGCC TGATTCGGGA GTTTACCCAG CGCCCCCTGCTAGTTGAGCG 29400 GGACAGGGGA CCCTGTGTTC TCACTGTGAT TTGCAACTGT CCTAACCTTGGATTACATCA 29460 AGATCTTTGT TGCCATCTCT GTGCTGAGTA TAATAAATAC AGAAATTAAAATATACTGGG 29520 GCTCCTATCG CCATCCTGTA AACGCCACCG TCTTCACCCG CCCAAGCAAACCAAGGCGAA 29580 CCTTACCTGG TACTTTTAAC ATCTCTCCCT CTGTGATTTA CAACAGTTTCAACCCAGACG 29640 GAGTGAGTCT ACGAGAGAAC CTCTCCGAGC TCAGCTACTC CATCAGAAAAAACACCACCC 29700 TCCTTACCTG CCGGGAACGT ACGAGTGCGT CACCGGCCGC TGCACCACACCTACCGCCTG 29760 ACCGTAAACC AGACTTTTTC CGGACAGACC TCAATAACTC TGTTTACCAGAACAGGAGGT 29820 GAGCTTAGAA AACCCTTAGG GTATTAGGCC AAAGGCGCAG CTACTGTGGGGTTTATGAAC 29880 AATTCAAGCA ACTCTACGGG CTATTCTAAT TCAGGTTTCT CTAGAATCGGGGTTGGGGTT 29940 ATTCTCTGTC TTGTGATTCT CTTTATTCTT ATACTAACGC TTCTCTGCCTAAGGCTCGCC 30000 GCCTGCTGTG TGCACATTTG CATTTATTGT CAGCTTTTTA AACGCTGGGGTCGCCACCCA 30060 AGATGATTAG GTACATAATC CTAGGTTTAC TCACCCTTGC GTCAGCCCACGGTACCACCC 30120 AAAAGGTGGA TTTTAAGGAG CCAGCCTGTA ATGTTACATT CGCAGCTGAAGCTAATGAGT 30180 GCACCACTCT TATAAAATGC ACCACAGAAC ATGAAAAGCT GCTTATTCGCCACAAAAACA 30240 AAATTGGCAA GTATGCTGTT TATGCTATTT GGCAGCCAGG TGACACTACAGAGTATAATG 30300 TTACAGTTTT CCAGGGTAAA AGTCATAAAA CTTTTATGTA TACTTTTCCATTTTATGAAA 30360 TGTGCGACAT TACCATGTAC ATGAGCAAAC AGTATAAGTT GTGGCCCCCACAAAATTGTG 30420 TGGAAAACAC TGGCACTTTC TGCTGCACTG CTATGCTAAT TACAGTGCTCGCTTTGGTCT 30480 GTACCCTACT CTATATTAAA TACAAAAGCA GACGCAGCTT TATTGAGGAAAAGAAAATGC 30540 CTTAATTTAC TAAGTTACAA AGCTAATGTC ACCACTAACT GCTTTACTCGCTGCTTGCAA 30600 AACAAATTCA AAAAGTTAGC ATTATAATTA GAATAGGATT TAAACCCCCCGGTCATTTCC 30660 TGCTCAATAC CATTCCCCTG AACAATTGAC TCTATGTGGG ATATGCTCCAGCGCTACAAC 30720 CTTGAAGTCA GGCTTCCTGG ATGTCAGCAT CTGACTTTGG CCAGCACCTGTCCCGCGGAT 30780 TTGTTCCAGT CCAACTACAG CGACCCACCC TAACAGAGAT GACCAACACAACCAACGCGG 30840 CCGCCGCTAC CGGACTTACA TCTACCACAA ATACACCCCA AGTTTCTGCCTTTGTCAATA 30900 ACTGGGATAA CTTGGGCATG TGGTGGTTCT CCATAGCGCT TATGTTTGTATGCCTTATTA 30960 TTATGTGGCT CATCTGCTGC CTAAAGCGCA AACGCGCCCG ACCACCCATCTATAGTCCCA 31020 TCATTGTGCT ACACCCAAAC AATGATGGAA TCCATAGATT GGACGGACTGAAACACATGT 31080 TCTTTTCTCT TACAGTATGA TTAAATGAGA CATGATTCCT CGAGTTTTTATATTACTGAC 31140 CCTTGTTGCG CTTTTTTGTG CGTGCTCCAC ATTGGCTGCG GTTTCTCACATCGAAGTAGA 31200 CTGCATTCCA GCCTTCACAG TCTATTTGCT TTACGGATTT GTCACCCTCACGCTCATCTG 31260 CAGCCTCATC ACTGTGGTCA TCGCCTTTAT CCAGTGCATT GACTGGGTCTGTGTGCGCTT 31320 TGCATATCTC AGACACCATC CCCAGTACAG GGACAGGACT ATAGCTGAGCTTCTTAGAAT 31380 TCTTTAATTA TGAAATTTAC TGTGACTTTT CTGCTGATTA TTTGCACCCTATCTGCGTTT 31440 TGTTCCCCGA CCTCCAAGCC TCAAAGACAT ATATCATGCA GATTCACTCGTATATGGAAT 31500 ATTCCAAGTT GCTACAATGA AAAAAGCGAT CTTTCCGAAG CCTGGTTATATGCAATCATC 31560 TCTGTTATGG TGTTCTGCAG TACCATCTTA GCCCTAGCTA TATATCCCTACCTTGACATT 31620 GGCTGGAAAC GAATAGATGC CATGAACCAC CCAACTTTCC CCGCGCCCGCTATGCTTCCA 31680 CTGCAACAAG TTGTTGCCGG CGGCTTTGTC CCAGCCAATC AGCCTCGCCCCACTTCTCCC 31740 ACCCCCACTG AAATCAGCTA CTTTAATCTA ACAGGAGGAG ATGACTGACACCCTAGATCT 31800 AGAAATGGAC GGAATTATTA CAGAGCAGCG CCTGCTAGAA AGACGCAGGGCAGCGGCCGA 31860 GCAACAGCGC ATGAATCAAG AGCTCCAAGA CATGGTTAAC TTGCACCAGTGCAAAAGGGG 31920 TATCTTTTGT CTGGTAAAGC AGGCCAAAGT CACCTACGAC AGTAATACCACCGGACACCG 31980 CCTTAGCTAC AAGTTGCCAA CCAAGCGTCA GAAATTGGTG GTCATGGTGGGAGAAAAGCC 32040 CATTACCATA ACTCAGCACT CGGTAGAAAC CGAAGGCTGC ATTCACTCACCTTGTCAAGG 32100 ACCTGAGGAT CTCTGCACCC TTATTAAGAC CCTGTGCGGT CTCAAAGATCTTATTCCCTT 32160 TAACTAATAA AAAAAAATAA TAAAGCATCA CTTACTTAAA ATCAGTTAGCAAATTTCTGT 32220 CCAGTTTATT CAGCAGCACC TCCTTGCCCT CCTCCCAGCT CTGGTATTGCAGCTTCCTCC 32280 TGGCTGCAAA CTTTCTCCAC AATCTAAATG GAATGTCAGT TTCCTCCTGTTCCTGTCCAT 32340 CCGCACCCAC TATCTTCATG TTGTTGCAGA TGAAGCGCGC AAGACCGTCTGAAGATACCT 32400 TCAACCCCGT GTATCCATAT GACACGGAAA CCGGTCCTCC AACTGTGCCTTTTCTTACTC 32460 CTCCCTTTGT ATCCCCCAAT GGGTTTCAAG AGAGTCCCCC TGGGGTACTCTCTTTGCGCC 32520 TATCCGAACC TCTAGTTACC TCCAATGGCA TGCTTGCGCT CAAAATGGGCAACGGCCTCT 32580 CTCTGGACGA GGCCGGCAAC CTTACCTCCC AAAATGTAAC CACTGTGAGCCCACCTCTCA 32640 AAAAAACCAA GTCAAACATA AACCTGGAAA TATCTGCACC CCTCACAGTTACCTCAGAAG 32700 CCCTAACTGT GGCTGCCGCC GCACCTCTAA TGGTCGCGGG CAACACACTCACCATGCAAT 32760 CACAGGCCCC GCTAACCGTG CACGACTCCA AACTTAGCAT TGCCACCCAAGGACCCCTCA 32820 CAGTGTCAGA AGGAAAGCTA GCCCTGCAAA CATCAGGCCC CCTCACCACCACCGATAGCA 32880 GTACCCTTAC TATCACTGCC TCACCCCCTC TAACTACTGC CACTGGTAGCTTGGGCATTG 32940 ACTTGAAAGA GCCCATTTAT ACACAAAATG GAAAACTAGG ACTAAAGTACGGGGCTCCTT 33000 TGCATGTAAC AGACGACCTA AACACTTTGA CCGTAGCAAC TGGTCCAGGTGTGACTATTA 33060 ATAATACTTC CTTGCAAACT AAAGTTACTG GAGCCTTGGG TTTTGATTCACAAGGCAATA 33120 TGCAACTTAA TGTAGCAGGA GGACTAAGGA TTGATTCTCA AAACAGACGCCTTATACTTG 33180 ATGTTAGTTA TCCGTTTGAT GCTCAAAACC AACTAAATCT AAGACTAGGACAGGGCCCTC 33240 TTTTTATAAA CTCAGCCCAC AACTTGGATA TTAACTACAA CAAAGGCCTTTACTTGTTTA 33300 CAGCTTCAAA CAATTCCAAA AAGCTTGAGG TTAACCTAAG CACTGCCAAGGGGTTGATGT 33360 TTGACGCTAC AGCCATAGCC ATTAATGCAG GAGATGGGCT TGAATTTGGTTCACCTAATG 33420 CACCAAACAC AAATCCCCTC AAAACAAAAA TTGGCCATGG CCTAGAATTTGATTCAAACA 33480 AGGCTATGGT TCCTAAACTA GGAACTGGCC TTAGTTTTGA CAGCACAGGTGCCATTACAG 33540 TAGGAAACAA AAATAATGAT AAGCTAACTT TGTGGACCAC ACCAGCTCCATCTCCTAACT 33600 GTAGACTAAA TGCAGAGAAA GATGCTAAAC TCACTTTGGT CTTAACAAAATGTGGCAGTC 33660 AAATACTTGC TACAGTTTCA GTTTTGGCTG TTAAAGGCAG TTTGGCTCCAATATCTGGAA 33720 CAGTTCAAAG TGCTCATCTT ATTATAAGAT TTGACGAAAA TGGAGTGCTACTAAACAATT 33780 CCTTCCTGGA CCCAGAATAT TGGAACTTTA GAAATGGAGA TCTTACTGAAGGCACAGCCT 33840 ATACAAACGC TGTTGGATTT ATGCCTAACC TATCAGCTTA TCCAAAATCTCACGGTAAAA 33900 CTGCCAAAAG TAACATTGTC AGTCAAGTTT ACTTAAACGG AGACAAAACTAAACCTGTAA 33960 CACTAACCAT TACACTAAAC GGTACACAGG AAACAGGAGA CACAACTCCAAGTGCATACT 34020 CTATGTCATT TTCATGGGAC TGGTCTGGCC ACAACTACAT TAATGAAATATTTGCCACAT 34080 CCTCTTACAC TTTTTCATAC ATTGCCCAAG AATAAAGAAT CGTTTGTGTTATGTTTCAAC 34140 GTGTTTATTT TTCAATTGCA GAAAATTTCA AGTCATTTTT CATTCAGTAGTATAGCCCCA 34200 CCACCACATA GCTTATACAG ATCACCGTAC CTTAATCAAA CTCACAGAACCCTAGTATTC 34260 AACCTGCCAC CTCCCTCCCA ACACACAGAG TACACAGTCC TTTCTCCCCGGCTGGCCTTA 34320 AAAAGCATCA TATCATGGGT AACAGACATA TTCTTAGGTG TTATATTCCACACGGTTTCC 34380 TGTCGAGCCA AACGCTCATC AGTGATATTA ATAAACTGGC GGCGATATAAAATGCAAGGT 34440 GCTGCTCAAA AAATCAGGCA AAGCCTCGCG CAAAAAAGAA AGCACATCGTAGTCATGCTC 34500 ATGCAGATAA AGGCAGGTAA GCTCCGGAAC CACCACAGAA AAAGACACCATTTTTCTCTC 34560 AAACATGTCT GCGGGTTTCT GCATAAACAC AAAATAAAAT AACAAAAAAACATTTAAACA 34620 TTAGAAGCCT GTCTTACAAC AGGAAAAACA ACCCTTATAA GCATAAGACGGACTACGGCC 34680 ATGCCGGCGT GACCGTAAAA AAACTGGTCA CCGTGATTAA AAAGCACCACCGACAGCTCC 34740 TCGGTCATGT CCGGAGTCAT AATGTAAGAC TCGGTAAACA CATCAGGTTGATTCATCGGT 34800 CAGTGCTAAA AAGCGACCGA AATAGCCCGG GGGAATACAT ACCCGCAGGCGTAGAGACAA 34860 CATTACAGCC CCCATAGGAG GTATAACAAA ATTAATAGGA GAGAAAAACACATAAACACC 34920 TGAAAAACCC TCCTGCCTAG GCAAAATAGC ACCCTCCCGC TCCAGAACAACATACAGCGC 34980 TTCACAGCGG CAGCCTAACA GTCAGCCTTA CCAGTAAAAA AGAAAACCTATTAAAAAAAC 35040 ACCACTCGAC ACGGCACCAG CTCAATCAGT CACAGTGTAA AAAAGGGCCAAGTGCAGAGC 35100 GAGTATATAT AGGACTAAAA AATGACGTAA CGGTTAAAGT CCACAAAAAACACCCAGAAA 35160 ACCGCACGCG AACCTACGCC CAGAAACGAA AGCCAAAAAA CCCACAACTTCCTCAAATCG 35220 TCACTTCCGT TTTCCCACGT TACGTAACTT CCCATTTTAA GAAAACTACAATTCCCAACA 35280 CATACAAGTT ACTCCGCCCT AAAACCTACG TCACCCGCCC CGTTCCCACGCCCCGCGCCA 35340 CGTCACAAAC TCCACCCCCT CATTATCATA TTGGCTTCAA TCCAAAATAAGGTATATTAT 35400 TGATGATG 35408

What is claimed is:
 1. A packaging cell line which expresses anadenovirus E1 gene and an adenovirus E4 gene or a functional fragment ofsaid E1 or E4 genes.
 2. A stable packaging cell which comprises: (a) afirst adenovirus DNA under the regulatory control of a first promoter,wherein said first adenovirus DNA encodes an adenovirus E1 protein; and(b) a second adenovirus DNA under the regulatory control of an induciblepromoter, wherein said second adenovirus DNA encodes an adenovirus E4ORF6 protein, wherein said packaging cell contains no other adenovirusE4 ORF DNA; and whereby, in the presence of an inducing agent, said E1and E4ORF6 proteins are expressed in amounts sufficient to permitinfection by, and recovery of, an adenovirus having functional deletionsin its E1 and E4 genes.
 3. The cell of claim 2, which is a human cell.4. The cell of claim 2 in which the inducing agent is a metal.
 5. Thecell of claim 2 in which the metal is zinc.
 6. The cell of claim 2 inwhich the inducing agent is a glucocorticoid.
 7. The cell of claim 2 inwhich the inducing agent is dexamethasone.
 8. The cell of claim 2 inwhich the first adenovirus DNA and the second adenovirus DNA are withinthe same nucleic acid molecule.
 9. The cell of claim 2 in which thenucleic acid expressing the adenovirus E4ORF6 protein is pMMTVE4ORF6.10. The cell of claim 2 in which the nucleic acid expressing theadenovirus E4ORF6 protein is pMTE4ORF6.
 11. The cell of claim 2 which is293 (MT-ORF6).
 12. The cell of claim 2 which is HeLa (MT-ORF6).